Scheme 23. 12 experimental materials for primary culture of adipocytes

DMEM-ADMEM-B collagenase male rats

Reagents and kits

KRBH buffer KRBH-A buffer

Instruments and consumables

Petri dish conical centrifugal tube polypropylene tube low density polypropylene bottle nylon filter dissection scissors Perry tweezers plastic box hay cutter pipette

experimental procedure

First, remove the epididymal fat pad.

1. When 70% carbon dioxide is charged? And 30% oxygen. Rats (Sprague-Dawley male rats: 145~ 170 g, CD series) were anesthetized in a plastic box with mixed gas.

2. Bleeding due to decapitation with a straw cutter.

3. Soak the mouse in 70% ethanol for a while.

4. Try to take out the epididymal fat pad under aseptic conditions.

(a) Cut the skin of the lower abdomen with a scalpel to expose the peritoneum.

(b) Cut the peritoneum with another dissecting scissors, and then pull the testis upward with Perry forceps.

(c) Incite the epididymal fat pad and pay attention to the preservation of blood vessels.

5. Transfer the tissue to the culture laboratory.

Digest and wash collagenase from adipocytes (add 20 mg/ml collagenase to 2 ml KRBH buffer and then filter with 0.22 micron filter).

6. put 4 g fat pads (equivalent to 8 epididymal fat pads) into 4 ml KRBH buffer (Krebs-Ringer solution, pH 7.4), and add 10 mmol/L NaHCO3, 30 mmol/L HEPES (Shima), 200 nmol/L adenosine (Boche) and/kloc-. When preparing 1 L KRBH buffer, 7 g NaCl, 0.55 g KH2PO4, 0.25 g MgSO4? 7H2O, 0.84 g NaHCO3, 0.11gcalcl2, 7. 15 g HEPES, 10 g BSA, 1 ml 200 μmol/L adenosine, and add ultrapure water to/kloc-0. Before use, heat it to 37°C and adjust the pH value to 7.4. ) (37℃), packed in a 30 ml low-density polypropylene bottle.

7. Cut the fat pad into tissue blocks with a diameter of about 2 mm.

8. Put the tissue block into the bottle, and then add 1 ml collagenase solution. Culture in a 37°C water bath oscillator for about 65438 0 hours until the cell mixture forms a creamy viscous liquid.

9. After collagenase digestion, add 4 ml KRBH buffer (37°C) to the bottle.

10. By stirring the cells in the mixing bottle, then gently filter the cells into a 50 ml conical centrifuge tube with a nylon screen with a pore size of 250 microns (placed in a support or funnel).

1 1. Add 30ml of KRBH buffer (37°C) to the centrifuge tube and wash the cells. Centrifuge for a short time (200 g) with a desktop centrifuge, and then suck out the supernatant with a straw. Note that fat cells float on top of the aqueous buffer.

12. wash the cells by adding 40 ml KRBH buffer, centrifuge and remove the supernatant. Repeat this step 1 time.

13. Use 40 ml of DMEM-A (DMEM, pH 7.4), add 25 mmol/L glucose, 2 mmol/L glutamine, 200 mmol/L (R)-N6-(L- methyl -2- phenylethyl) adenosine (PIA, Sigma) and 66. According to 100 ml, heat to 37 C (37 C), and clean the battery twice before use.

14. Suspend floating cells with about 40% cytokine DMEM-A.

Second, primary culture of adipocytes.

15. transfer 2ml of 40% cytocrit DMEM-a suspension into 60 mm culture dish with 200 μl large-caliber pipette.

16. Place the Petri dish in a humid incubator with 37℃ and 5% CO2. Under the condition of 1.5 h

17. Add 5 ml of DMEM-B (DMEM-A, 7% bovine serum albumin) to the Petri dish.

At this time, glucose uptake test [Quom 1998] should be carried out to check the cell viability.

-

Journal of sun yat-sen university of medical sciences (Acadjsum), 200 1, 22 (6): 443 ~ 446443.

Primary culture of human preadipocytes

1, 2, Li Yan 2, Yang 1, Kuang 1.

(Sun Yat-sen University of Medical Sciences 1. Department of Obstetrics and Gynecology, Sun Yixian Memorial Hospital, 2. Department of Biology, Guangzhou, Guangdong 5 10 120)

Objective To establish a primary culture method of human preadipocytes in order to further study the biological characteristics of human adipose tissue hyperplasia. Methods Adult abdominal adipose tissue was selected and prismatic cells were cultured by primary digestive cell culture. At the same time, skin tissue culture was taken as control. Results The cultured prismatic cells had uniform composition, vigorous proliferation and high differentiation rate. Through the observation of morphological dynamic changes, growth curve and oil red O fat staining extraction method, it was proved that it was an active preadipocyte and the whole process of its proliferation was reproduced in vitro. Conclusion There are preadipocytes in mature adipose tissue that can differentiate and mature and produce fat. Because adipocyte is one of the classic target cells of insulin action, this experiment laid a foundation for further study on obesity and insulin resistance-related diseases such as polycystic ovary syndrome.

Key words: preadipocyte; Cell culture; fat tissue

Classification number of China Library: Q254, R7 1 1.75 Document ID: A document number:1000-257x (2001) 06-0443-04.

primarycultureofhumanpredipocyte

Wang Zhuchen 1, Liu Jianzhong 2, Li Yan 2, Yang Dongzi 1, Kuang Jianquan 1

(1. Department of Obstetrics and Gynecology, Zhongshan Memorial Hospital, 2. Biology department,

Medical College of Sun Yat-sen University, Guangzhou, China, 5 10 120)

Abstract: Objective To establish a culture method of primary human preadipocytes in order to better understand the characteristics of human preadipocytes. Methods Fibroid cells were cultured by primary cell culture. Results Fibroblasts had high uniformity, proliferation and high differentiation rate. Their dynamic morphological changes, growth curves, extraction of staidintractopsiclippid with oil redo, all verified their irpreipoxyidentity. Under controlled conditions, preipocytes replay hyperplasmia process invitro. Conclusion There are preadipocytes and differentiated adipocytes in adult adipose tissue. Because of the classic target cell insulin, such as adipocytes, this study is called TheBasisforFurtherProbingToObesity and Sullinsistance, Design, Base, Diagnosis and Syndrome Syndrome.

Key words: preadipocyte; Cell culture; fat tissue

Adipocyte is one of the classic target cells of insulin action, and

Obesity and insulin resistance related diseases, such as polycystic ovary syndrome.

Research is closely related, and it has received more attention in the field of gynecological endocrinology in recent years.

See. Preadipocytes are a kind of cells that can proliferate and differentiate into adipocytes.

Chemotactic specialized precursor cells, whose existence and function continue.

In human life, we have cultivated preadipocytes from human adipose tissue.

Cells laid the foundation for further research.

Date of receipt: 200 1-04-23

Fund Project: Supported by Scientific Research Fund of Guangdong Provincial Health Department (200 1 189)

,; 1 materials and methods 1. 1 tissues were from normal-weight patients who underwent fallopian tube recanalization in the gynecological endocrine ward of our hospital from September 2000 to June 2006. Age 20~40 years old, healthy, no other acute or chronic diseases. Subcutaneous adipose tissue and skin tissue were obtained during laparotomy.

444

1.2 test material

1.2. 1 main inspection supplies and chemical reagents DMEM/F 12 medium (1! 1), mycoplasma-free fetal bovine serum, collagenase A, Hepes, human transferrin, penicillin and streptomycin were all purchased from GIBCO Company. Biotin, pantothenate, hydrocortisone, triiodothyronine (T3) and 3 isobutyl 1 methylxanthine are all products of SIGMA. Bovine serum albumin was purchased from Roche. The inorganic reagents needed for the experiment were purchased from the Chemical Testing Wholesale Department of Guangzhou Pharmaceutical Company, all of which were analytical pure reagents. Culture bottles were purchased from KORNING Company.

1.2.2 preparation of culture medium and main reagents A[ 1] Journal of Sun Yat-sen University of Medical Sciences (Acadjsum), 200 1, 22 (6) culture medium A was used to prepare cell suspension, which was inoculated in a 25cm2 culture bottle with a density of 3? 65,438+00 cells were incubated for 65,438+06h in 37# CO2 incubator with 5% volume fraction. After that, PBS gently washed away the non-adhered substances in the culture bottle, and induced and maintained the differentiation of adipocytes with medium C. After 3 days, it was changed to B medium, and then it was changed to B medium every 2 ~ 3 days. 1.3.2 histological staining cut the cover glass of human preadipocytes and skin fibroblasts into 0.5 cm? 10cm in size, placed in a culture bottle when inoculated, and taken out every 2 or 3 days after the cells adhered to the wall for staining. Immerse the upward side of fat cells in the volume fraction.

Fix 10min in isotonic saline buffer numbered 10% formaldehyde, then put it in PBS buffer, gently rinse for a while, stand upright to make the residual water flow to the edge, and blot it with absorbent paper. After a little drying, human preadipocytes were cultured with Sudan red%&; The nucleus was stained by drop staining and double staining with Melhematoxylin, and sealed with glycerol gelatin. Skin fibroblasts were stained with hematoxylin eosin, dehydrated and transparent, sealed with neutral resin, observed by light microscope and photographed to save the results.

1.3.3 Draw the cell growth curve. Inoculate the cell suspension into 24 culture bottles, and randomly divide them into 8 groups with 3 bottles in each group. The total number of cells in each bottle in a group was detected every 2 days, and the average value of 3 bottles was taken, so that the eighth group ended. Cell counting method refers to medical cell biology experiment [3].

1.3.4 The extraction method of oil red O is the same as that of 1.3.3. According to Ramirez[2]4, 5g DMEM/f 12 culture powder was taken according to the instructions, and fetal bovine serum was added to make its volume fraction 10%, and the volume was fixed to 500mL with triple distilled water. Medium b: take 10g DMEM/f 12 culture powder according to the instructions, and the final concentrations are 15mmol/LNAHCO3, 15mmol/LHEPES, 33mol/L pantothenic acid and 17mol/L human transformation respectively. 100nmol/L hydrocortisone, 60nmol/L insulin, 02nmol/LT3, triple distilled water to1000 ml; Medium c: take 200mL of medium a, and add 3 isobutyl 1 methylxanthine to make its final concentration be 0.25mmol/l; Digestive juice: weigh collagenase (? Type) 150mg, bovine serum albumin 2g, 00 1mol/L phosphate buffered saline solution (PBS) to100ml; Erythrocyte lysis buffer: weigh appropriate amount of NH4Cl, K2HPO4 and EDTA, and use triple distilled water to make their final concentrations 154mmol/L, 57mmol/L and 0 1mmol/L respectively.

Fix the volume to 250 ml. After adjusting the pH value of the above solutions to 74 ~ 76,022 022m, they were filtered and sterilized with microporous membrane under positive pressure, and then packaged and stored in -30#. Oil red o working fluid [2], etc. The culture was fixed with isotonic saline buffer with the volume fraction of 10% formaldehyde 1h, and then washed with distilled water. Suck 10mL oil red o working solution, put the culture surface down, and pour out the oil red o in the bottle after 2 hours.

Rinse the culture bottle several times with working solution and distilled water until it floats completely. Put the dyed culture bottle into 32# incubator to evaporate the water in the bottle, then add 1mL isopropanol, immediately suck the extracted dye solution with a straw, and measure the absorbance with HITACHIG2000 spectrophotometer at the wavelength of 5 10nm. : Weigh 42g oil red O, dissolve it in 1200mL isopropanol, and let it stand overnight at room temperature. After filtering with analytical filter paper, collect the filtrate, add 900mL of tri-distilled water, let it stand overnight at No.4 position again, filter it twice again, and store it at room temperature. 1.2.3 instrument CO2 incubator, inverted microscope, etc. 1.3 method

1.3. 1 Primary culture of human preadipocytes and skin fibroblasts [1] About 60g of adipose tissue and 05cm of skin tissue were cut during the operation. 3cm was used for fibroblast culture as control. PBS washing, separation and removal of visible fiber components and blood vessels in adipose tissue, skin tissue needs to remove epidermis and subcutaneous fat, cut into about 2mm? 2mm small pieces, add digestive juice, and put them in a 37# CO2 incubator with a volume fraction of 5% for digestion. 15h later, 200? G Short-term centrifugation, removing floating adipocytes and culture medium, preparing the precipitated cells into cell suspension with erythrocyte lysis buffer again, and incubating with 37# 10min to remove contaminated erythrocytes. 1502 Results 2. 1 Morphological observation of primary cultured human preadipocytes and skin fibroblasts showed that the cells were initially round after adhering to the wall. The nuclear/plasma ratio is relatively large (figure 1 1), the cells gradually become spindle-shaped in 4 days, and the prismatic cells proliferate in about 7 days, and the fat particles begin to accumulate after the area reaches the bottom of the culture bottle 1/2 (figure 12). On the 9th day, it was observed that the cells were gradually transformed from edges to ellipses or circles (Figure 13), and fat particles were accumulated.

Wang Zhuchen et al. primarily cultured 445 adipocytes from human preadipocytes (Figure 14). Adipocytes cultured in vitro have large volume, thin cell membrane, unsmooth, uneven and wrinkled membrane contour. There are a large number of round lipid droplets in the cytoplasm, which vary in size and are mostly scattered. There are also cases where a large number of small lipid droplets contact to form large lipid droplets, and the nucleus is still at the edge. At the same time, the cultured skin fibroblasts have similar proliferation rate, but they are always long prismatic and have no lipid droplet accumulation (Figure 2 1, Figure 22).

2.2 Growth curve of human preadipocytes and skin fibroblasts

It can be seen from the growth curve that the doubling time of human preadipocytes (Figure 3 1) and skin fibroblasts (Figure 32) is 60h and 55h, respectively.

. To explore the significance of 3. 1 human preadipocyte culture in gynecological endocrine field. Adipocyte is an important part of adipose tissue and the main body of its function. Contemporary studies believe that fat cells in adipose tissue are very active, and the number, morphology and intracellular fat content of cells are in dynamic changes and remain for life. Obesity is considered to be the result of uncontrolled proliferation and differentiation of fat cells. At present, fat

Obesity has become a disease closely related to gynecological endocrine. Obesity can produce insulin resistance/hyperinsulinemia, which is the same danger signal of many clinical diseases or symptoms, especially common endocrine and metabolic diseases such as diabetes, hypertension and atherosclerosis, so it has become a research hotspot of equal interest in many medical disciplines in recent years. Especially in recent years, research has found that

Fig. 3. 1 human preadipocyte growth curve

Fig. 3. 1 human preadipocyte growth curve

Cultured insulin resistance/hyperinsulinemia is also related to hyperandrogenism and anovulation in women of childbearing age. Polycystic ovary syndrome,

About 5%~ 10% of women of childbearing age suffer from this disease, often accompanied by obesity, and the cause is unknown. At present, it is considered that polycystic ovary syndrome is an endocrine and metabolic disease characterized by insulin resistance, and hyperinsulinemia secondary to insulin resistance plays an important role in causing hyperandrogenism and infertility [4]. Adipocytes act as insulin inhibitors.

Fig. 3.2 Growth curve of human skin fibroblasts

Figure 3.2 Human skin is one of the sensitive target cells, and it is of special clinical significance to study its potential insulin resistance mechanism. Preadipocyte culture in vitro

It is an ideal model to understand the whole process of adipose tissue occurrence and proliferation, directly observe the regulation of various factors on this process and study its mechanism. Although 3T3L 1, 3T3F442A, ob 17 series of cell lines derived from mouse preadipocytes have been established abroad, so far, no human preadipocyte line has been established, which limits further research.

3.2 Biological characteristics of human preadipocytes cultured in vitro

At present, there are two kinds of preadipocyte culture systems at home and abroad. One is derived from vascular stromal cells (SVF), which is a typical preadipocyte. Van et al. [6] systematically studied SVF and formed a complete preadipocyte theory. They treated the cut adipose tissue with collagenase, then centrifuged the tissue suspension, in which the vascular component of the precipitated matrix was SVF, and cultured SVF. Compared with cultured fibroblasts, the proliferation rate of the two cultures is the same, and the doubling time is about 55 hours. Comparing the cells under the microscope, it was found that after 9 days of culture before 2.3, the fat content in adipocytes increased rapidly, reaching the peak at around 16d, while only a small amount of fat accumulated in skin fibroblasts (Figure 4).

. Fig. 4 changes of intracellular fat content during the growth of preadipocytes and skin fibroblasts (fig. 4) changes of adipocytes and skin fibroblasts.

446

A large number of lipid droplets can be accumulated in the cytoplasm of fibroblasts, but there are no or only a few lipid droplets. This proves that SVF is a preadipocyte with the potential of proliferation and differentiation into adipocytes. The results of this experimental study are consistent with them and with the three standards proposed in reference [6]. In addition, in recent years, it has also been reported that the method of combining adipose tissue mass with ceiling culture has also been successful [7]. The preadipocytes obtained by this method are different from SVF, and may come from the cell islands found in mature adipocytes by Carraro et al. However, this method is cultivated in serum and incubated with mature adipocytes, which is not suitable for the in vitro research model of some cytokines secreted by mature adipocytes.

The culture of preadipocytes clarifies the relationship between proliferation and differentiation, which is currently considered as growth arrest? Grow again? The continuous relationship of cell differentiation. Growth pause is a necessary first step. After that, these cells have to undergo at least one division before continuing to transform into mature fat cells. As time goes by, the late marker of glycerol 3 phosphate dehydrogenase (GPDH)mRNA appears, which is also the rate-limiting enzyme necessary for fat synthesis and eventually becomes an adipocyte. Our study also observed that cells began to grow after a period of incubation, proliferated in about 3 or 5 days, and began to accumulate fat particles after 1 week. After 2 weeks of culture, adipocytes differentiated into lipid droplets or lipid droplets, which was consistent with the literature reports.

Oil red O staining extraction method can simply and quickly quantify the transformation rate of adipocytes before in vitro culture. The accuracy and sensitivity reported in the literature are similar to GPDH, which is a marker enzyme in the process of determining preadipocyte differentiation [2]. Therefore, it is often used to identify preadipocytes cultured in vitro. Under the action of insulin and hydrocortisone, the GPDH of preadipocytes cultured in vitro began to rise and increased rapidly, reaching the peak in about 8 days and maintaining at a high level [2]. We used oil red O dyeing extraction method to study. The results showed that preadipocytes began to accumulate fat on the third day of culture, and the accumulation amount increased obviously after 1 week, reaching the peak at the second week, which was consistent with the appearance of fat particles in cells after morphological culture 1 week. It also shows that the appearance of enzyme comes first and the appearance of fat comes later, and the increase of intracellular fat content is about 1 week later than that of GPDH, which is consistent with the literature report [2].

3.3 Characteristics of human preadipocyte culture technology

Adipose tissue is derived from primitive reticular connective tissue and consists of connective tissue and adipose cells. Adipocytes are the main cellular components, in addition to reticular cells, mesenchymal cells, tissue cells, endothelial cells and so on. Both adipocytes and fibroblasts are from mesoderm, so the cultured adipocytes and fibroblasts are similar [9], and the external culture conditions are different [10] 20065438,22 (6). The difference is that they need the action of adipogenic factors. At present, these substances are called insulin, glucocorticoid and glucocorticoid. Generally, DMEM and F 12 are used as culture medium, and the ratio is 1! 1 ratio, the number of cells is moderate, the cell adhesion is not very firm at the initial inoculation, and the movement should be gentle to avoid cell floating. The younger the selected material, the easier it will grow. Humans generally choose surgery to cut abdominal adipose tissue. If it is sucked with a syringe, it is easy to damage cells and reduce the survival rate. (See Figure 2 for figures 1 and 2 in this article) References: [1] Haunerh, Petruschketh, Russm, etal. Effectsovumornecrossis factor alpha (TNF) on glucose metabolism and lipid metabolism of new differentiated diabetes mellitus [J]. Diabetes mellitus,1995,38 (7): 764. [2]RamirezZacariasJL，CastroMunozledoF，Kuriharuchw。 Quantitative analysis of fatty transformation and triglyceride by oil staining [J]. Histochemistry,1992,97 (6): 493. [3] Zhao Gang and Liu Jianzhong. Experiments and exercises of medical cell biology [M]. Beijing: Science Press, 1999.35. [4] Du Naifa. Insulin and hepatopathy syndrome [J]. Endocrine and metabolin,1999,28 (2): 341. [5] Major Billings. Historical Review and Present Situation Review [J].PlastReconstrSurg,1989,83 (2): 368. [6] Van Erle, Bayless Sey, Roncari Ducker. Cytological and Enzymatic Characteristics of Adult Adipocyte Precursor Cell Culture [J].JClinInvest,1976,58 (9): 699. [7] Zhu Xiaohai, He Qinglian, Lin Zihao. Establishment of human preadipocyte culture, proliferation and differentiation model [J]. China Journal of Plastic Burn Surgery, 1999, 15 (3): 6544. Johnson Je Isletsoft preadipocytes attach great importance to today's discrimination [J].Celtissres, 199 1, 264 (2): 243. [9] Deng. Endothelium-smooth muscle co-culture: the effect of intercellular interaction on tissue plasminogen activator [J]. Journal of Sun Yat-sen Medical University, 1992, 13 (4): 18. [10] Zhu Yongyuan, Li Tianzeng, Su Aiyun, etc