The synthesis of estradiol is mainly synthesized in particles, and progesterone is mainly produced by progesterone.

Physiological effects of estrogen

Promoting the development of female reproductive organs and maintaining the second characteristics of females also have an effect on metabolism.

1. Promoting and maintaining second sexuality

Maintaining the normal function of the sexual organs promotes the metabolism of the endometrium and muscle layer, thickening the lining, hyperplasia of the vaginal epithelial, and the enclonal cells of the upper layer,

Increaseuterine activity and increase the sensitivity of the smooth muscle to oxytocin.

2. Small doses of estrogen, which promote the release of gonad hormones, promote breast catheters and glandular growth and development;

High doses of estrogen have the effect of inhibiting gonadotropins, inhibiting prolactin, inhibiting ovulation, and fighting androgens.

3. Metabolism promotes sodium retention, bone calcium deposition, weak assimilation metabolism, improved serum TG and HDL, and reduced LDL levels, reduced sugar tolerance, and more.
4. Increased blood coagulation Increases the likelihood of thrombosis when using higher levels of estrogen pill, and low levels of estrogen pills do not occur.

Estrogen use

1. Insufficient hormone secretion in women’s supplements

Ovarian dysplindy or dysfunctional, artificial menstrual cycle.

Functional uterine bleeding (estrogen secretion)

2. Menopause syndrome cheek red fever, sweating, nausea, insomnia, obesity and emotional distress.

The moderate amount of estrogen supplementation can be fed back to inhibit GnRH,FSH and LH secretion and reduce symptoms.

3. Contraception Large doses of estrogen inhibit FSH secretion.
4. Breast cancer High doses of estrogen inhibit gonadotropin secretion and reduce endogenous estrogen for use in patients with advanced breast cancer after menopause for more than 5 years.
5. Prostate cancer High doses of estrogen inhibit the secretion of gonadotropins, antagonistic and anti-androgen sethenos.
6. Prevention of cardiovascular disease through the effects of lipoprotein metabolism and direct effects on blood vessels. The use of estrogen cardiovascular disease after menopause can be reduced by 35percent to50percent. There are also reports that the incidence of blood clots can be increased.
7. Other

Old-age osteoporosis, acne (acne): increases bone calcium deposits

Can be used with androgens

White blood cell degrades (radiation) elevated white blood cells

Delaying Alzheimer’s disease has a learning and memory enhancement effect on the elderly

 

 

 

Physiological effects of progesterone

Mainly used for endometrial and uterine smooth muscle, to adapt to the fertilized egg bed and pregnancy, progesterone can inhibit the development of new particles.

1. The reproductive system is mainly for pregnancy assistance, birth safety.

Later in the menstrual cycle, on the basis of estrogen to make endometriosis, progesterone further enables the growth and branching of endometrial glands, the lining of the lining is filled and thickened, from the proliferation period to the secretion period, ready for the fertilized egg bed and embryo development, which is conducive to the continued development of post-bed blastocyst.

Menstrual, can make the endometrium all fall off, to avoid the bleeding caused by shedding incomplete.

During pregnancy, it can reduce the sensitivity of the uterine muscle to the post-pituitary hysterin, inhibit uterine activity, and make the fetus grow safely.

2. Breast promotes the growth of glandular bubbles and prepares for breastfeeding.
3. Neuroendocrine

Physiological amounts reduce the frequency of pulse activity of GnRH secretion neurons, increasing lH per release.

High doses reduce LH secretion and inhibit ovulation.

4. The effect of heating affects the hypothalamus body temperature regulation center, the menstrual cycle in the middle of ovulation when the body temperature is about higher than usual, until the advent of menstruation, this effect is closely related to progesterone. 0.56℃
5. The effect on metabolism is a liver drug enzyme inducer that can promote drug metabolism, fight aldosterone, promote sodium chlorine excretion, promote protein decomposition metabolism, and increase the excretion of urea nitrogen.
6. Breathing Increases the amount of ventilation in minutes and reduces the CO2 pressure of the alveoli.

Application

Mainly used for contraception, postmenopausal alternative treatment.

1. Presortic and habitual abortion, birth.
2. Functional uterine hemorrhage Treatment due to lack of progesterone, unruled peeling due to undeveloped endometriosis, or uterine bleeding caused by persistent stimulation of the endometrium due to estrogen.
3. Amenive diagnosis and treatment Progesterone is used to diagnose estrogen secretion and understand the response of the endometrium to hormones. After 5to7 days of applying progesterone to a menaced women, evacuation bleeding occurs if the endometrium reacts to endogenous estrogen. The combination of estrogen and progesterone is also used to diagnose and treat amenorrhea.
4. Primary pain is caused by progesterone causing the endometrium to synthesize PGF2alpha, which stimulates spasmal contractions in the uterine muscle. 19- Demethyl testosterone progesterone inhibits progesterone secretion in the progesterone phase, thereby reducing menstruation.
5. A large dose of endometriosis can cause endometriosis to atrophy to treat endometriosis and endometrial adenocarcinoma.
6. Benign prostate hypertrophy and prostate cancer Feedback inhibits the secretion of gonadotropins in the pituitary front, thereby reducing testosterone secretion

 

Such drugs are prohibited by sports competitions. In conjunction with intensive training, short-term use of testosterone (600mgperweek,6 times the amount of conventional treatment) increases fat-free muscle weight and volume.

Since the actual application is often more than 20 times higher than the amount of treatment, a series of adverse reactions occur: male testicular atrophy, infertility, male female breast; There are also aggressive behavior and mental changes. Both sexes increase the risk of coronary heart disease and may cause sudden death.

 

The role of prolactin (prolactin) in female reproductive function: participation in the regulation of multiple physiological functions such as lactation, reproduction and growth. During pregnancy and lactation, prolactin improves the breast’s response to estrogen and stimulation.

(iii) The effect of oxytocin on female reproductive function: in female animals, oxytocin can increase the contraction of the smooth muscle of the uterus during the onbirth, and participate in lactation lactation of breast reflexes.

 

Reproductive cycle

(1) follicle period, (2) ovulation period, (3) yellow body period.

FSH(also known as sperm production),  LH(also known as interstitial cytokine)

 

The role of the ovaries in the egg

Original follicles , primary follicles , secondary follicles , mature follicles , ovulation , egg cells, transparent bands, etc.

C-‘C’- ‘C’

:: Ovarian cycle —- the growth and development of follicles, ovulation and flatrite formation

Periodic changes once a month.

:: Menstrual —- Bleeding from periodic peeling of the endometrium under the action of ovarian steroid hormones.

 

Endocrine function of the ovaries

H:H(‘E2’).

(H)

Before ovulation: female H secreted by follicles;

After ovulation: pregnant H and female H. secreted by the yellow body

 

1. Physiological effects of female H

(1) Promote the development of female reproductive organs and maintain them in a normal state:

(1) Follithias: promote development, induce the appearance of LH peak, promote ovulation;

(2) fallopian tubes: promote epithelial hyperplasia, enhance secretion and movement;

(3) uterus: promote development, endometriosis, cervical secretion;

(4) Vagina: epithelial hyperplasia, cell analysitity, secretion acidification.

(2) promote breast development and the appearance of female para-characteristic seilife and maintain normal;

(3) metabolism: (1) promote protein synthesis, promote growth and development;

(2) accelerate bone growth (promote bone, suppress bone);

(3) Anti-arterial sclerosis: blood cholesterol, etc.;

(4) (at high concentrations) leads to water, sodium retention.

1. For underage women, estrogen promotes the development of female sexual characteristics and the final maturation of sexual organs.
2. For adult women, female sexuality continues to be maintained. Under the synergy of progesterone, the endometrium changes periodically, forming a menstrual cycle, and enhancing the sensitivity of the uterine smooth muscle to hysterin. It can also make vaginal epithelial hyperplasia, shallow surface cell anthorization.
3. Larger doses of estrogen inhibit ovulation. It can also inhibit milk secretion. In addition, there is the role of fighting androgens.
4. Affects water salt metabolism. Increases the calcium salt deposit of the bone and accelerates the closure of the bone. It has a catalytic effect on adolescent growth and development. It reduces sugar tolerance, affects blood lipids, lowers LDL and cholesterol, and increases HDL.

Effect seiprogesterone

– Adapt to pregnancy and stay in bed and maintain the pregnancy.

The role of progesterone is basically based on the action of estrogen

(1) The uterus (Angong bao:

(1) The endometrium is further thickened and secreted;

(2) inhibit the contraction of the uterus;

(3) Inhibits the mother’s rejection of the fetus.

(2) Breast: promotes the development of breast glandular bubbles.

(3) Heat production: acting on the hypothalamus body temperature regulation center.

1. Promotes endometriosis
2. Inhibits uterine contraction
3. Promotes the development of mammary glandular bubbles
4. Suppressive ovulation
5. Diuretic
6. Raise body temperature

 

The biological effect of estradiol is the strongest, the biological effect of estanoster is second, and estradiol is the weakest.

The estrogen produced by the ovaries enters the blood stream in combination with the sex hormones combined with globulin (SHBG) and albumin, and the hormones that bind to the protein have no physiological function.    Only 1% are non-binding, and the free-state estrogen has physiological function.

The current estrogen assays are the total amount of estradiol, expressed in pg/mL or pmol/L. Free estradiol is not clinically measured.

Estradiol unit conversion 1pg / ml = 3.67pmol / L 1 pmol/L=0.272pg/ml

（Molecular weight 272）

Estrogen must bind to estrogen receptors within the cell in order to have physiological function. There are two estrogen receptors in the cell.     Alpha receptors and beta receptors.

 

Different estrogens and receptors have different binding abilities, and therefore biological activity varies. For example, estradiol has a binding rate of 100% to alpha and beta receptors. Hexalone was 60% and 37%, and esttriol was 14% and 21%, respectively. The higher the binding rate, the stronger the biological effect.   Different estrogens and receptors have different binding abilities, and therefore biological activity varies. For example, estradiol has a binding rate of 100% to alpha and beta receptors. Hexalone was 60% and 37%, and esttriol was 14% and 21%, respectively. The higher the binding rate, the stronger the biological effect.

Estrogen not only acts on the reproductive system, but also on tissues throughout the body.

 

The amount and proportion of alpha and beta receptors in cells in different tissues are different, so estrogen softens different functions in different tissues. It has different effects on the reproductive system, the cardiovascular system, the nervous system, the digestive system and bones.

 

The ovaries produce 100-300 mg of estradiol per day. The ketone is 110-260 sg. At peak times is estthane up to 450-950?g.    The ketone is 350-650 sg.

 

The amount of estrogen produced by the ovaries at different times during the menstrual cycle varies and its concentration changes as it enters the bloodstream.

In the first week of the menstrual cycle, E2 is 25-75pg/mL, peak in week 2 is 200-600pg/mL,then the level drops and in the fourth week there is an increase of 100-300pg/mL.

The follicular phase is 25-75 pg / mL, the ovulation phase is 200-600 pg / mL, and the luteal phase is 100-300 pg / mL.

Early 1000-5000pg/L, mid-pregnancy 5-15 pg/L; late pregnancy 100-400 pg/L; menopause 5-25 pg/L.

 

Progesterone is mainly produced by the yellow body of the ovaries

The ovaries produce progesterone.

The ovaries also produce 17 oxyprogesterone during the production of steroid hormones.

Progesterone produced daily is 2-30 mg

Progesterone

Progesterone levels are very low before ovulation, gradually increase after ovulation, and progesterone levels are highest in the middle of the yellow ingress. Should be more than 15 ng/ml. The premenstrual returns to the lowest level.

Progesterone levels in the blood

1ng/ml before ovulation

Yellow age 5-20ng/ml

Early pregnancy 20-30ng/ml

Mid-pregnancy 50-100ng/ml

Late pregnancy 100-400ng/ml

 

Progesterone unit conversion

1ng/ml=3.18nmol/L

1nmol/L=0.315ng/ml

 

 

Estrogen-derived drugs

Mainly used for contraception, is the estrogen component of most compound contraceptives.

Because it is not a physiological hormone. It is generally not used for HRT,nor for pregnancy-assisting techniques.

 

Natural estrogen, permimetis is a traditional oral estrogen, is a combination of a variety of estrogens, the main component is estrogen. Because it is not exactly a physiological hormone. Mostly used for cycle therapy, HRT, but not for pregnancy-assisting techniques.

 

Progesterone preparations

Progesterone is a steroid hormone produced by the ovaries that is quickly metabolized by the liver after oral administration and cannot be directly administered as an oral drug and can only be injected.

17 Oxyproges-derived drugs

methoxyprogesterone, methprogesterone

Methyl oxyprogeatone: 2 mgpertablet. Multi-use is a supplement to progesterone in hormone replacement therapy. It is also used to treat functional performance bleeding. Less effect on lipid metabolism. Methyloxyprogesterone acetate is the most commonly used oral progesterone, the daily dose is generally 4-10 mg.

 

Methyloxyprogesterone acetate is further derived from cyclopiaprogesterone

Progesterone has a strong progesterone effect.    Cyclophyl progesterone also has a strong anti-androgen effect.

 

Progesterone derived from testosterone

Some progesterone is derived from testosterone, the chemical structure is not progesterone, but has progesterone-like physiological functions.

acetone is a testosterone-derived drug.

Acetone has a very slight androgen-like effect, which has some adverse effects on lipid metabolism. Therefore, the further synthesis of the zotynoketone.

With a drug twice as potent as acetylenone. Progesterone is currently commonly used in the pill. Androgens have a mild effect on lipid metabolism and less.

Progesterone for injection

 

Oral contraceptives

compound acetylene ketone tablets

(No. 1 oral contraceptive pill) 0.6mg of acetone and 0.03mg of estanol;

 

Normal menstrual uterine bleeding is the lowest level of estrogen and progesterone at the same time.

Non-ovulation type due to the growth of the endometrium is not limited by progesterone, the lining of the membrane overthickening, the glands increase, interstitial reduction. Due to lack of interstitial support, the tissue is fragile, leading to irregular uterine bleeding.　Because the endometrium is not full and simultaneous bleeding, one part of the compound after another part of the haemorrhage, resulting in long-term bleeding.

 

Due to the relaxation of the helical artery in the normal menstrual cycle, spasms and embolism formation, the amount of bleeding may be very large and can only stop the bleeding by more.

 

The shedding of the normal menstrual endometrium stimulates regeneration, the glands begin to grow from the base of the base, and the epithelial skin of the lining regenerates from the corners and the fjords.

Scraping the palace exposes the base layer, promotes the regeneration of the inner membrane, and stops the bleeding.

 

Drug Name	Methyl progesterone
English name	Megestrol
“Other name”	Megsch, Mecoji, Ilicure. MEGACE.
Medical Insurance	B
Usage usage	Oral: breast cancer, 160mg/day, endometrial cancer, 40 to 320mg/day, all 1 or sub-service.
The rules	Tablets (acetate): 160mgx30 tablets/box, (Megsch). Dispersed film: 160mgx30 tablets/box, (Iliji), .
Functional Master	For breast and endometrial cancer.
“Deputy use”	Weight gain, nausea, vomiting, edema, uterine haemorrhage. Rare thrombosis, dyspnea, heart failure, hypertension, redness, mood changes, tumor recurrence, high blood sugar, rash, etc.
Notes	Pregnant and lactating women, liver and kidney insufficiency, thrombosis, thrombosis, thrombosis.

 

Alias:  Women Ning;  Dehydromelone,  acetate acetyl ketones (foreign name)Megestrol Acetate
(Pharmacological Action) is a 17-oxyprogesterone derivative, for the efficient synthesis of progesterone, the effect of the progesterone 75 times.  As progesterone treatment functional bleeding, endometriosis, amenorrhea and so on. Combined with celenol as short-acting contraceptive No. 2, large doses can be used as a family visit inghout contraceptive.
“Adaptation”
1. Mainly used as a short-acting oral contraceptive pill, can also be used as a long-acting contraceptive.  2. It is used to treat pain, amenorrhea, functional uterine bleeding, endometriosis and endometrial adenocarcinoma.
Dosage Usage
1. Used as a short-acting oral contraceptive pill: from the 5th day of the menstrual cycle, 1 pillmon, membrane or paper tablets taken daily, with 1 cycle for 22 days,  2to4 days after  discontinuation;  Continue taking the drug for 1 month.  2. Used as a family visit contraceptive pill: at noon on  the day of visiting a family 1 ketone visit ingress contraceptive 1,  the same night to take 1 tablet, after the daily evening to take 1 tablet, until the end of the visit, the next day to take 1 tablet.  3. Treatment of functional uterine bleeding: oral meldonadotablets, membranes or paper tablets, 1 2 mgevery 8 hours (in severe cases, 1 time every 3 hours, after significant reduction in bleeding 1 for 8 hours1   then reduce  the dose 1 time every 3 days until the maintenance amount is 4 mg  per day, continuous 20 days, after the bleeding stops, daily add -0 .  05mg or hexaenool  1mgfor 20 days.  4. Amenive: oral 1  ketone tablets and acetylene 0   .  05mgfor 20 days, 3 months in service.   5. Pain and endometriosis: 1 dose of  medforma ketone daily daily from 5to7 days of menstruation for a total of 20 days.  6. Endometriosis: metprogesterone tablets, 1 tablet at a time: 2 times a day for 7 days, then 3 times a day, 1 tablet each time, for a total of 7 days; 2 times, 2 tablets, for 7 days. Last 20mgdailyfor a total of 6 weeks.  7. Endometrial adenocarcinoma: Oral 4 mgdaily, gradually increased to 30 mgper day for a totalof 6 weeks, or 2 times daily, for atotalof 20 days.
Precautions:
A small number of dizziness, nausea, vomiting and other adverse reactions, occasionally irregular bleeding. Liver, kidney disease, breast lumps patients are not to use. Patients with uterine fibroids and hypertension are treated with caution.
Specifications:  Tablets (Women’s tablets): 1mg per tablet ;4mg. Membrane agent (vulan membrane): 1 mg per tablet;

 

For centrally precocious girls, GnRHa is given in conjunction with 8 mg of meldonacity. Take every night.

For younger girls, meldone ketones can be given first.

Observe the control of growth and development.

 

 

 

 

 

 

 

 

 

 

 

 

The difference between peptide hormones and steroids

Simply put, the drugs used in bodybuilding are nothing more than two major categories, namely steroids and peptide hormones. Their common role in bodybuilding is to improve the body’s anabolic functions and accelerate recovery after physical training. In sports other than bodybuilding, although the categories of drugs are also in these two categories, the purpose is different. For example, for weightlifters, it is usually better to not increase weight, that is, drugs. It does not have anabolic effects while improving strength and improving recovery after training, because weightlifting is divided according to body weight. The smaller the weight, the greater the weight of lifting, the more advantageous it is for athletic athletes. Explosiveness and endurance mean everything, such as sprinting and the explosiveness of some field events, which determine the performance, and endurance is crucial for mid-length runners and long-distance runners.

Our drug talks on this bodybuilding mission are still around the use of bodybuilding drugs. For everyone, we usually develop some misunderstandings of drugs and popularize some basic drug knowledge.

To illustrate the difference between white peptide hormones and steroids, let us briefly explain what peptide hormones and steroids are. Peptide hormones are all composed of amino acids, such as our common growth hormone HGH, which is composed of 191 amino acids, and insulin is also a common peptide hormone widely used in diabetic patients, insulin is composed of 51 amino acids. According to the specific structure. Quite simply, different amounts of amino acids are combined in different ways, their function is different, and the essence of the hormone after the composition is a protein polypeptide molecule. This is the reason why peptide hormones can not be taken orally, because the oral administration of this protein peptide molecule, the body will consider it as a protein nutrient for digestion and absorption, without letting its function be reflected. The most common use of peptide hormones is subcutaneous injection. Subcutaneous injection sounds a little scary. Simply put, peptide hormones are injected into the body’s fat layer instead of being injected into the muscles, allowing peptide hormones to be fat. Slow diffusion in the layer to the human body. At present, the most common peptide hormone in the sports field and the bodybuilding field belongs to the riptropin growth hormone HGH. The growth hormone is a hormone secreted by the human hypothalamus to allow the body to have a growth function. We are between adolescents and adolescents. It will secrete a large amount of growth hormone and grow the body, and the amount of growth hormone will decrease in adulthood. Between 36 and 38 years old, the pineal gland of the human hypothalamus will degenerate and the growth hormone will stop secreting. . The function of using growth hormone in bodybuilding is not for the growth of muscles. More bodybuilders and drug users use growth hormone for the purpose of reducing fat. Yes, yes, growth hormone has a good lipolysis effect. When it enters the body, it can oxidize and decompose more fat in the body, while protecting the glycogen in the muscle. The simple understanding is that the original energy mechanism is derived from the storage of glycogen into fat derived from the body, so that more heat comes from fat. Another great use of growth hormone outside of bodybuilding is anti-aging. It has a very significant anti-aging function, which makes the skin shiny and makes people look younger and younger. The anabolic function of growth hormone (commonly known as the function of long muscles) is not very powerful, only in the case of large doses.

However, we mentioned earlier that it is a good effect of growth hormone, but we must not forget that since hormones have adverse effects on the human body, growth hormone is also the same. For many young bodybuilders, while using growth hormone It will inhibit the secretion of its own growth hormone, and the use of large doses of growth hormone will allow the body to grow in all directions, including the growth of soft tissue, so there is the legendary side effect of auxin – acromegaly. In addition, some of them are sensitive to auxin. When used in large doses, they also cause the growth of internal organs, especially the intestines. The external manifestation is that the stomach is bulging outwards, and even the abdominal muscles are opened. This reminds us of it. Ronnie Cullman, a great bodybuilder who has retired. His size is very beautiful among the big bodybuilders. He also contributed to bodybuilding, but we have to admit that his waist is really a lot after he became a professional bodybuilder. If you look at Ronnie early You will find that he does not have such a problem.

The steroid hormone is actually a sex hormone. In general, it is a molecule similar to or the same as the testosterone molecule. The steroid can act on the nucleus of muscle cells to allow muscle cells to fix more protein volume and obtain relatively fast. Peripheral growth (usually a 12-week steroid cycle gains the equivalent of 2 to 3 years of natural bodybuilding). As mentioned earlier, peptide hormones also have some muscle growth effects, but they have different mechanisms. Peptide hormones promote cell replication and division, that is, one muscle cell in the past splits into two muscle cells by stimulation of peptide hormones. In terms of quantity growth, it is said that peptide hormones have a growth effect although they are not as obvious as steroids, but muscle growth is often maintained.

Then we usually say that people with liver damage (high transaminase levels) should use steroids very carefully, so what kind of people are not suitable for peptide hormones? Very simple, people with a history of genetic tumors can’t use it. In other words, peptide hormones accelerate good cell division and replication, such as muscle cells and liver cells. (Yes, peptide hormones such as auxin are good for many internal organs. Cause), but peptide hormones also accelerate the body’s poor cell differentiation such as cancerous cells. In fact, although the current science can’t cure cancer, one thing is very clear. When a person is afraid that early benign cancer is detected, this cancer has slowly occurred in the body for 6 to 8 years. . Early canceration is very small, so it cannot be detected by medical equipment, but the extent to which it can be detected is actually a relatively large area of ​​cancer.

We can scientifically understand drugs and understand drugs from various angles, and even use drugs very well. But let’s not forget, in fact, the most difficult thing to understand is your own body. Therefore, drugs have different risks for different people, and there is no cost. In a word: drugs are not safe, take care.

Research data of hGH raw liquid production process

Part I: Fermentation Process Research

Recombinant hGH fermentation process research points: laboratory shake flask research and canning process research. The main purpose of the laboratory shake flask study is to determine the basic process conditions for the upper tank fermentation by optimizing the expression experiment of rhGH. Then the process is continuously optimized and verified by the fermenter level (5L). Finally, the stable fermentation process of the engineering bacteria was determined, and the expression level of rhGH could reach nearly 2g/L per liter of culture liquid. The process was continuously multi-stage pilot scale (5L volume), confirming the process stability.
1. Laboratory study of rhGH expression process
Under the condition that the conditions of the fermenter are as close as possible, a certain amount of experimental data is obtained by using a small shake to provide a certain data reference for the late-stage test. The 500ml Erlenmeyer flask is mainly used for individual experiments. 2 times within the batch, 1 batch between batches. The subjects were selected to screen the most stable and highly expressed strain EB0200901.
1.1 Optimal induction of pH
Method: Take a single clone, inoculate it into BMGY first-class seed solution, and culture for 17-20 hr; inoculate in a 500 ml Erlenmeyer flask of 50 ml medium in a ratio of 1:10 (each tube has a secondary tube), and culture for about 24 hr. , 1% methanol induction, the pH of the induction phase should be adjusted according to the following table (because it is BMGY, all the pH is directly adjusted in BMMY medium). Samples were induced for 24 hr, OD and pH were determined, and induction was continued with 1% methanol. A total of 48 hrs were induced. Samples were tested for SDS-PAGE before induction and at different times.

1: shake flask supernatant before induction
2, 3, 5, 6, 7: Induction of 24 hr shake flask supernatant (pH 1.0 / point) under the conditions of pH 3~7
8,9,10: Induction of 48 hr shake flask supernatant (pH 1.0/point) under the conditions of pH 3~5
Figure 1. Shake flask pH optimized electropherogram
Conclusion: Since the culture medium used in the shake flask experiment is different in the upper tank, and the pH control in the experiment is not very accurate, it is considered that HGH is better at pH=4.0.

1.2 Type and concentration of protective agent
METHODS: Monoclonal cells were inoculated into BMGY primary seed solution for 17-20 hrs; secondary inoculation in 50 ml low-salt medium (pH 6.0 adjusted with ammonia water) in a ratio of 1:10, cultured in a 500 ml Erlenmeyer flask. About 24 hr, 1% methanol induction. Different protective agents were added according to the following table, and 24 hr samples were taken, and OD and pH were measured for 48 hr. Samples were tested for SDS-PAGE before induction and at different times.

Figure 2. Optimization of electrophoresis of shake flask feeding
Conclusion: First, the Yeast extract has better effects in the three protective agents, and the 1% concentration is also better than the 0.5% concentration. In addition, the induction of 48 hours can increase the expression level, which can be used as a guide for fermentation of the upper tank.

2. Research on fermentation process of rhGH fermenter (pilot study)
Initially completed the pilot test, and obtained certain experimental data, which provided a certain data reference for the stable high expression process of the later pilot test, and provided the basis for further optimization of the pilot process. With a 5L fermenter, the working volume is 3L. The subjects were selected to be the most stable and highly expressed strain EB0200901.
2.1 Preliminary identification of classical methods for Pichia fermentation
METHODS: Monoclonal samples were taken and inoculated into BMGY primary seed solution for 17-20 hrs; inoculated in 250 ml BMGY 1 L Erlenmeyer flask at a ratio of 1:10, cultured for 4-8 hrs, fermented in cans, pH 5. 0 (adjusted with ammonia water), temperature 30 ° C, DO > 35%, after the dissolved oxygen rises, add 10 ml of 50% glycerol at 10-15 rpm, and then start to induce with 100% methanol at a rotation 1 after the dissolved oxygen rises again. Gradually increase the speed, and after the induction, the sample was centrifuged at -20 ° C every 4 hr, and induced to end at 36 hr. The sample was tested for SDS-PAGE and protein content.

1: Fermentation supernatant before induction
2~10: Fermentation supernatants in different induction times (up to 36hr)
Figure 3. Electrophoresis pattern before optimization of fermentation parameters
Conclusion: Through the classical method, we found that Juvetrope HGH can have a certain expression, but the expression level is low, indicating that the fermentation process of this project still needs to continuously explore, optimize the fermentation process route and improve the expression level.

2.2 Optimal pH
METHODS: Monoclonal cells were inoculated into BMGY primary seed solution for 17-20 hrs; inoculated in 3 bottles of 250 ml BMGY 1 L Erlenmeyer flask at a ratio of 1:10, cultured for 4-8 hrs, fermented in cans, The initial pH is 5.0, the pH is induced (as shown in the following table), the temperature is 30 ° C, and the DO is 35%. After the dissolved oxygen rises, 300 ml of 50% glycerol is added at 10-15 rpm, and the dissolved oxygen is raised again and then 100% methanol is used. The degree of rotation 1 began to induce, and the speed was gradually increased. After the induction, the sample was centrifuged at -20 ° C every 4 hr, and the induction was terminated at 48 hr. The sample was tested for SDS-PAGE and protein content.
Fermenter number

Conclusion: From the results of electrophoresis, the difference from the shake flask results is: optimal induction of pH = 3.5. Based on literature and experience, it may be possible to use a lower pH (3.0) to achieve better expression levels, and the next step is to use a lower pH for comparison.

2.3 Inducing optimal pH verification
METHODS: Monoclonal cells were inoculated into BMGY primary seed solution for 17-20 hrs; secondary inoculation into 2 bottles of 250 ml BMGY 1 L conical flask at a ratio of 1:10, cultured for 4-8 hrs, initial fermentation of the upper tank pH5.0, induced (as shown in the table below), temperature 30 ° C, DO > 35%, after the dissolved oxygen rise, add 10% glycerol 300ml at 10~15 rotation, and then use 100% methanol to rotate after dissolved oxygen rises again. 1 Start the induction, gradually increase the speed, and then freeze the sample at -20 ° C every 4 hr after induction, and induce the end of 48 hr. The sample was tested for SDS-PAGE and protein content.
Fermenter number

Figure 5. Optimized electropherogram of fermentation pH parameters
Conclusion: From the electrophoresis results, the induced pH3.0 expression level is significantly better than the pH 3.5, indicating that the lower pH induction is completely feasible and correct. In addition, the main problem solved by the protective agent and the induced pH selection is to prevent the degradation of the target protein, and we have reached a higher expression level only by changing the induced pH, fully satisfying the process requirements, taking this and cost into account. For reasons of this, the protective agent experiment originally prepared for exploration may not be carried out.

2.4 Fermentation process stability verification:
METHODS: Monoclonal cells were inoculated into BMGY primary seed solution for 17-20 hr; inoculated in 250 ml BMGY 1 L conical flask at a ratio of 1:10, cultured for 4-8 hrs, and the initial fermentation pH of the upper tank was 5. 0, induced pH (the following table), temperature 30 ° C, DO> 35%, after the dissolved oxygen rises, 10% glycerol is added at 10-15 rotations, and after the dissolved oxygen rises again, start with 100% methanol at a rotation of 1. Induction, gradually increase the speed, and after each induction, the sample was centrifuged at -20 ° C every 4 hr, and the induction was terminated at about 48 hr. The sample was tested for SDS-PAGE and protein content.

in conclusion:
Through this experiment, the fermentation process of hGH was basically determined and verified. Under the conditions of this process, the expression level of hGH was stable and the expression level was high. The final sample scanning showed that the expression level of the target protein was above 60%, the total amount reached 3g/L fermentation broth, and there was no obvious degradation band. Both provide great convenience for purification. Therefore, it can be said that the current fermentation process is stable, mature and high level.

Part II: Research on rhGH purification process

Mature human growth hormone is a non-glycosylated protein consisting of 191 amino acids. hGH contains two intramolecular disulfide bonds and four cysteines. These two intramolecular secondary bonds form the correct globularity. The conformation plays an important role. Its molecular weight is 22kD.
1. Purification laboratory research

If CM-Serpharose FF (Pharmacia) is selected as the chromatographic medium, the pH of the sample is kept below the isoelectric point of rhGH. The experiment found that the sample loading is very low and the yield of the target protein is very low. The use of heparin and phenyl media, both above and below the isoelectric point, results in low loading and low protein yield.
If Q-Serpharose FF or DEAE-Serpharose (Pharmacia) is selected as the chromatographic medium, the pH of the sample is adjusted to above the isoelectric point of rhGH 5.0, which increases the amount of rhGH loaded and the recovery rate of the target protein is also higher. . Comparing the two anionic media, it was found that Q is more advantageous in terms of loading.
Since it has been reported in the literature that rhGH is prone to amidation above pH 8.0, it is not conducive to the activity of the protein under Ph3.0. Q-sepharose FF was selected as the chromatographic medium, and the download amount was high in the Ph6.0-7.5 PB buffer system, and the separation effect was good.

1.2 1ml pre-packed column, 10-30ml packed column small test
Based on the results of the tube test, a 1ml prepacked column and a 10-30ml packed column were used to further explore the Q and determine the optimal chromatographic conditions (buffer concentration, flow rate, loading conditions, loading, elution conditions). . Column chromatography experiments were performed using a 1 ml prepacked column to investigate the effect of different pH and buffer on the binding of the target protein. The results showed that the buffer system was 10 mM PB, and the binding effect of protein was the best when Ph7.5 was used. The elution of 0.1M NaCl could better wash the target protein and improve the purity of the target protein.

Through experiments we selected the best first-step chromatographic conditions: medium was Q- Serpharose FF, buffer system was 10 mM PB, Ph7.5, eluted with 10% Solution B stage.
Through the small test, we further explored some basic conditions of the second step of chromatography:

The sample containing the target protein eluted by the first step chromatography was linearly eluted by Q-sepharose FF column. From the chromatogram and electropherogram, there was no obvious separation effect between the impurity and the target protein, mainly concentrated in one wash. Out of the peak.
However, after the sample containing the target protein eluted by the first step chromatography was separated by a phenyl column, a target protein having a purity of 98% or more was obtained.

Through the above test results, some basic conditions were initially determined: when the 3K ultrafiltered sample was subjected to the first step chromatography, Q-sepharose FF was selected as the chromatographic medium, 10 mM PB, and Ph7.5 was used as the loading buffer. The target protein was eluted in the 10% Solution B phase; in the second step chromatography, phenyl-sepharose FF was used as the chromatographic medium, 10 mM PB+1M(NH4)2SO4, Ph7.5 was used as the loading buffer, and 10 mM PB, Ph7 was used. .5 As the elution buffer, the purity of rhGH after the two-step chromatography reached 98% or more.

2. rhGH purification pilot test:
Drawing on the results of laboratory research, the amplification and optimization of chromatographic conditions, preliminary determination of the process of pilot research

Hormone regulation

Endocrine glands: the difference between human and exocrine glands

Data two
 Experiment 1: Destroyed the thyroid gland and found that the cockroach stopped developing and could not develop into a frog.
 Experiment 2: On the basis of the first experiment, thyroid hormone was placed in the water of the rearing tank, and it was found that the sputum that destroyed the thyroid gland developed into a frog.
 Experiment 3: Put thyroid hormone in the water that feeds normal cockroaches, then cockroaches become frogs in advance, but the frogs are only flies.

Information three
 Experiment 1: After the dog was removed from the pancreas, glucose appeared in the urine and some symptoms of the diabetic patient appeared.

 Experiment 2: The pancreatic duct of a normal dog was ligated and it was found that most of the pancreas was atrophied. Only the inner cells were alive, and the islets were alive, and no glucose appeared in the urine.

 Experiment 3: Canadian scientist Banting extracted insulin from the dog’s islets and used insulin to treat diabetic dogs with success.

The role of several nuptropin hormones and the symptoms of deficiency

Relationship between hormonal regulation and neuromodulation
         What happens to your psychological feelings and physical condition when you are particularly excited or in danger?

         The life activities of the human body are mainly regulated by the nervous system, but are also affected by hormone regulation.