【摘要】：臨床上因創(chuàng)傷、腫瘤術(shù)后、感染及先天缺陷的患者，需要大量的脂肪組織來(lái)修復(fù)軟組織缺損。然而，常用的自體脂肪移植有很多的局限。脂肪組織工程是應(yīng)用干細(xì)胞對(duì)軟組織進(jìn)行重建的一門(mén)新興技術(shù)，干細(xì)胞和成脂因子在其中起著關(guān)鍵的作用。脂肪干細(xì)胞由于其來(lái)源廣泛、含量豐富、取材方便、具有多向方分化潛能，已廣泛應(yīng)用于組織工程研究。脂肪組織不僅僅是能量存儲(chǔ)的器官，并且能分泌大量細(xì)胞因子。我們?cè)谥窘M織塊原代法培養(yǎng)脂肪細(xì)胞時(shí)發(fā)現(xiàn)，脂肪干細(xì)胞向脂肪細(xì)胞方向分化。脂肪組織分泌物可能含有，能誘導(dǎo)脂肪干細(xì)胞脂向分化的成脂因子�，F(xiàn)在研究比較成熟的體外成脂誘導(dǎo)因子包括：胰島素、吲哚美辛等，對(duì)生理性成脂因子的研究較少。本課題采用組織塊原代培養(yǎng)法獲取脂肪干細(xì)胞，探索脂肪組織分泌物與成脂間的關(guān)系，及脂肪組織分泌物中可能的成脂因子。對(duì)生理性成脂因子的研究將為脂肪組織工程應(yīng)用于臨床奠定基礎(chǔ)。 方法： 1、脂肪組織塊原代培養(yǎng)法分離培養(yǎng)大鼠脂肪干細(xì)胞，采用倒置顯微鏡觀察細(xì)胞的形態(tài)和生長(zhǎng)狀況。將原代細(xì)胞培養(yǎng)傳代，純化至第四代后，分別用成脂、成骨、成神經(jīng)化學(xué)誘導(dǎo)劑進(jìn)行誘導(dǎo)培養(yǎng)，然后進(jìn)行油紅O染色、茜素紅染色及熒光免疫實(shí)驗(yàn)。 2、脂肪組織塊貼壁法收集前三天培養(yǎng)液作為ATE，對(duì)第四代生長(zhǎng)狀態(tài)良好的大鼠脂肪干細(xì)胞，進(jìn)行誘導(dǎo)。而后進(jìn)行油紅O染色實(shí)驗(yàn)。 3、用含有FBS和無(wú)FBS的培養(yǎng)基對(duì)脂肪組織塊進(jìn)行培養(yǎng)并收集ATE，分別對(duì)大鼠脂肪干細(xì)胞進(jìn)行成脂誘導(dǎo)，進(jìn)行油紅O染色實(shí)驗(yàn)。 4、收集無(wú)FBS培養(yǎng)基獲取的ATE，分為100μg/ml、250μg/ml、500μg/ml組分，對(duì)第四代大鼠脂肪干細(xì)胞進(jìn)行誘導(dǎo)，分別觀察各誘導(dǎo)組成脂情況，進(jìn)行油紅O染色實(shí)驗(yàn)、成脂率檢測(cè)實(shí)驗(yàn)，及細(xì)胞增殖和遷移實(shí)驗(yàn)。 5、用超濾管將ATE按分子量富集為5個(gè)不同的組分，分別對(duì)第四代脂肪干細(xì)胞進(jìn)行誘導(dǎo)，觀察。并進(jìn)行western blot實(shí)驗(yàn)，熒光免疫染色實(shí)驗(yàn)。 6、以脂肪干細(xì)胞分泌蛋白作為對(duì)照，將脂肪組織分泌物進(jìn)行質(zhì)譜分析，通過(guò)信息學(xué)分析探索ATE中的成脂因子。并用western blot實(shí)驗(yàn)驗(yàn)證。 結(jié)果： 1、脂肪組織塊法獲得的細(xì)胞生長(zhǎng)狀態(tài)良好，能向脂肪細(xì)胞、成骨細(xì)胞及神經(jīng)細(xì)胞系方向分化。 2、ATE誘導(dǎo)二天時(shí)，可見(jiàn)個(gè)別第四代大鼠脂肪干細(xì)胞胞漿內(nèi)有脂滴形成，第七天時(shí)，脂滴融合變大。陰性對(duì)照組未見(jiàn)脂滴形成。 3、獲取ATE時(shí)添加或不添加FBS，在第七天時(shí)ADSCs胞漿中均有成熟脂滴形成，兩組成脂率無(wú)統(tǒng)計(jì)學(xué)差別(P0.05)。 4、500μg/ml組分較100μg/ml、250μg/ml組分成脂率高。不同濃度的ATE對(duì)大鼠脂肪干細(xì)胞的增殖均有抑制作用，但是各組間抑制程度相仿，無(wú)統(tǒng)計(jì)學(xué)意義(P0.01)。ATE對(duì)大鼠脂肪干細(xì)胞的遷移沒(méi)有影響。 5、分子量100KDa組分誘導(dǎo)大鼠脂肪干細(xì)胞，可見(jiàn)細(xì)胞脂向分化，而其它組未見(jiàn)成脂。western blot，熒光免疫實(shí)驗(yàn)結(jié)果表明分子量100KDa誘導(dǎo)組細(xì)胞,成脂相關(guān)蛋白表達(dá)陽(yáng)性。 6、通過(guò)質(zhì)譜分析找出差異表達(dá)蛋白中，分子量大于100KDa的蛋白有Col I、CP、IGg2a、FN，western bolt實(shí)驗(yàn)再次驗(yàn)證結(jié)果的可靠性。 結(jié)論： 1、脂肪組織塊法獲得的細(xì)胞有脂肪干細(xì)胞的生物學(xué)特性，具有良好的多向分化能力。 2、ATE能誘導(dǎo)脂肪干細(xì)胞成脂分化。 3、FBS對(duì)ATE獲取的成脂能力無(wú)影響。 4、濃度較高的500μg/ml組較濃度較低組的成脂能力更強(qiáng)。ATE能夠抑制細(xì)胞增殖，與干細(xì)胞成脂分化機(jī)制相符，但這種抑制不是無(wú)限制的。ATE對(duì)細(xì)胞遷移無(wú)影響。 5、脂肪組織塊分泌物中具有成脂分化能力的因子集中在分子量100KDa組分，為進(jìn)一步研究新的生理性的成脂因子奠定了基礎(chǔ)。 6、通過(guò)我們的實(shí)驗(yàn)證實(shí)，，Col I、CP、IGg2a、FN的確存在于脂肪組織分泌物中，其表達(dá)量及種類(lèi)與單純的脂肪干細(xì)胞分泌蛋白不同，是可能的成脂因子。
[Abstract]:A large number of adipose tissues are needed to repair soft tissue defects in patients with trauma, tumor surgery, infection and congenital defects. However, the commonly used autologous fat transplantation has many limitations. Adipose tissue engineering is a new technique for the application of stem cells to the reconstruction of soft tissue. Stem cells and lipid factors play a key role in it. Adipose stem cells are widely used in tissue engineering research. Adipose tissue is not only an organ of energy storage, but also a large number of cytokines. We found fat stem cells in fat tissue blocks and found fat stem cells to fat. The secretions of adipose tissue may be contained in the adipose tissue, which can induce fat stem cell fat to differentiate into fat forming factors. Now the mature in vitro lipid inducing factors include insulin, indomethacin, and less research on physiological lipid factors. The relationship between the secretions of the adipose tissue and the fat formation and the possible lipid factors in the secretions of the adipose tissue. The study of the physiological lipid factors will lay the foundation for the clinical application of adipose tissue engineering.
Method:
1, the fat tissue block was used to culture the rat fat stem cells by primary culture, and the morphology and growth status of the cells were observed by inverted microscope. The original cells were cultured and purified to fourth generations. The cells were induced and cultured with fat, bone and neurochemical inducers respectively. Then the oil red O staining, alizarin red staining and fluorescence immunization were carried out. Test.
2, the fat tissue block adherence method was used to collect the culture medium for the first three days as ATE to induce the fat stem cells of the fourth generation healthy rats, and then the oil red O staining experiment was carried out.
3, the fat tissue block was cultured and ATE was collected with the medium containing FBS and no FBS, and the fat stem cells of the rat were induced and the oil red O staining experiment was carried out.
4, the ATE obtained from the FBS medium was collected, which was divided into 100 g/ml, 250 g/ml and 500 mu g/ml components. The fourth generation of rat fat stem cells were induced, respectively, to observe the induced composition of the lipids, the oil red O staining experiment, the test of the fat percentage and the proliferation and migration of the cells.
5, the molecular weight of ATE was enriched into 5 different components by the ultrafiltration tube, and the fourth generation of fat stem cells were induced and observed respectively. The Western blot experiment and the fluorescence immunostaining experiment were carried out.
6, using the secretory protein of adipose stem cells as the control, the fat tissue secretions were analyzed by mass spectrometry, and the lipid forming factors in ATE were explored by informatics analysis. The results were verified by Western blot test.
Result:
1. Adipose tissue block method can induce cell growth in good condition and differentiate into adipocytes, osteoblasts and neural cell lines.
2, when ATE was induced for two days, the lipid droplets in the fat stem cells of the fourth generation rats were found to form, and the lipid droplets became larger at the seventh day. No lipid droplets were formed in the negative control group.
3. Added or not added FBS to ATE, mature lipid droplets were formed in the cytoplasm of ADSCs on the seventh day, and there was no significant difference between the two groups (P 0.05).
The 4500 u g/ml components were 100 g/ml and 250 micron g/ml group had high fat percentage. Different concentrations of ATE had inhibitory effect on the proliferation of rat fat stem cells, but the degree of inhibition was similar, and there was no statistical significance (P0.01).ATE had no effect on the migration of fat stem cells in rats.
5, the molecular weight 100KDa component induced rat fat stem cells, which showed that the cell fat was differentiated, but the other groups did not have fat.Western blot. The results of fluorescence immunoassay showed that the molecular weight of 100KDa induced group cells, and the expression of lipid related protein was positive.
6. Mass spectrometry analysis showed that the proteins with molecular weight greater than 100KDa were ColI, CP, IGg2a, FN and Western bolt.
Conclusion:
1. The cells obtained by adipose tissue block method have the biological characteristics of adipose-derived stem cells and have good multidirectional differentiation ability.
2, ATE can induce adipose differentiation of fat stem cells.
3, FBS had no effect on the fat forming ability of ATE.
4, the higher concentration of 500 mu g/ml group was stronger than the lower concentration group and.ATE could inhibit cell proliferation, which was consistent with the mechanism of lipid differentiation of stem cells, but this inhibition was not unrestricted.ATE on cell migration.
5, the factor of lipid differentiation in the secretions of fat tissue is concentrated in the molecular weight 100KDa component, which lays the foundation for further study of the new physiological fat forming factors.
6, our experiments show that Col I, CP, IGg2a, FN do exist in the secretions of adipose tissue, and the amount and type of expression are different from those of pure adipose stem cells, which are possible lipid factors.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2013
【分類(lèi)號(hào)】：R329

【共引文獻(xiàn)】

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