【摘要】： 目的:采用酶預(yù)消化后連續(xù)組織塊法進(jìn)行大鼠脂肪來源干細(xì)胞的體外培養(yǎng),并與膠原酶消化法的培養(yǎng)效果相比較,研究脂肪來源干細(xì)胞的獲取途徑并為其體外增殖培養(yǎng)方法提供參考依據(jù)。 方法:取一月齡SD大鼠腹股溝處脂肪組織,分A、B兩組。A組使用D-Hank's液沖洗凈后剪成8mm~3左右組織塊。將組織塊置于尼龍網(wǎng)上放至培養(yǎng)皿中,0.25%胰蛋白酶消化5min,0.1%Ⅰ型膠原酶消化組織塊20min,棄去消化液體,加入培養(yǎng)基后放于孵化箱中繼續(xù)培養(yǎng)。2～3天后將組織塊連同尼龍網(wǎng)一起拎除放至下一培養(yǎng)皿中以連續(xù)組織塊法培養(yǎng)3～4次。B組使用膠原酶消化法培養(yǎng)。采用MTT檢測方法對(duì)細(xì)胞增殖活性進(jìn)行比較,流式細(xì)胞儀檢測脂肪干細(xì)胞表面標(biāo)志物。 結(jié)果:A、B兩組培養(yǎng)的細(xì)胞均具有典型的干細(xì)胞形態(tài)特征,生長曲線相近,脂肪干細(xì)胞標(biāo)志物CD29、CD44、CD54、CD105呈陽性表達(dá),造血干細(xì)胞標(biāo)志物CD34、CD45陰性表達(dá),骨髓來源干細(xì)胞標(biāo)志物CD106陰性表達(dá)。A組的陽性表達(dá)率略高于B組,兩組間的陽性表達(dá)率無統(tǒng)計(jì)學(xué)上的差異(P＞0.05)。 結(jié)論:酶預(yù)消化連續(xù)組織塊法是進(jìn)行脂肪來源干細(xì)胞體外培養(yǎng)的另一種途徑,獲得脂肪來源干細(xì)胞的純度略高于酶原消化法。優(yōu)點(diǎn)是能短時(shí)間內(nèi)獲得多個(gè)脂肪干細(xì)胞樣本,對(duì)細(xì)胞損傷較小,具有一定的實(shí)驗(yàn)應(yīng)用價(jià)值。不足之處是細(xì)胞起始濃度較低,生長周期略長。 目的:探討SD大鼠脂肪來源干細(xì)胞(ADSCs)和雪旺細(xì)胞(Schwann,SC)利用Transwell培養(yǎng)板共培養(yǎng)時(shí),ADSCs的生長增殖特性以及其向神經(jīng)細(xì)胞分化的能力,并和使用試劑誘導(dǎo)(BME+BHA)下的ADSCs生長結(jié)果相比較,為構(gòu)建組織工程人工神經(jīng)提供種子細(xì)胞來源的理論和依據(jù)。 方法:按實(shí)驗(yàn)第一部分獲取ADSCs,同時(shí)取大鼠坐骨神經(jīng)按植塊法獲取雪旺細(xì)胞。取P3代脂肪來源干細(xì)胞分三組,A組使用ADSCs和雪旺細(xì)胞置于Transwell培養(yǎng)板中構(gòu)建共培養(yǎng)體系,B組利用β-巰基乙醇(BME)+丁酸酯羥基茴香醚(BHA)作為ADSCs誘導(dǎo)因子使其向神經(jīng)細(xì)胞方向誘導(dǎo),C組為空白對(duì)照組。比較各組細(xì)胞形態(tài),觀察脂肪來源干細(xì)胞生長、軸突長度、NF-M和GFAP表達(dá)情況,并做統(tǒng)計(jì)學(xué)分析。 結(jié)果:A、B兩組ADSCs均有部分細(xì)胞分化出細(xì)長突起,細(xì)胞生長代謝旺盛,GFAP免疫熒光染色陰性、NF-M免疫熒光染色陽性。在細(xì)胞數(shù)量上,A組與C組無差異,但明顯高于B組(P＜0.05);在突起長度上,A組與B組無差異,但明顯高于C組(P＜0.05)。 結(jié)論:雪旺細(xì)胞與ADSCs共培養(yǎng)能促進(jìn)ADSCs向神經(jīng)細(xì)胞方向分化,誘導(dǎo)效果與使用BME+BHA試劑誘導(dǎo)的結(jié)果相近,并能有效防止ADSCs損傷和死亡,提示雪旺細(xì)胞和ADSCs共培養(yǎng)可以作為一種將ADSCs向神經(jīng)細(xì)胞方向誘導(dǎo)的方法在組織工程領(lǐng)域加以利用。
[Abstract]:OBJECTIVE: To compare the effect of collagenase digestion on adipose-derived stem cells (ADSCs) cultured in vitro by enzymatic pre-digestion and continuous tissue block method, and to study the ways of obtaining ADSCs and provide reference for the methods of proliferation and culture in vitro.
Methods: One month old SD rats were divided into two groups: group A and group B. Group A was washed with D-Hank's solution and cut into about 8 mm~3 pieces of tissues. The pieces were placed on nylon mesh and digested with 0.25% trypsin for 5 min and 0.1% collagenase type I for 20 min. The digested tissues were discarded and put into incubator after adding the medium. After 2-3 days, the tissues were removed together with nylon mesh and placed in the next culture dish for 3-4 times. Group B was cultured with collagenase digestion method. Cell proliferation activity was compared by MTT assay, and surface markers of adipose-derived stem cells were detected by flow cytometry.
Results: The cells cultured in A and B groups had typical morphological characteristics of stem cells, and the growth curves were similar. Adipose stem cell markers CD29, CD44, CD54 and CD105 were positive, hematopoietic stem cell markers CD34 and CD45 were negative, and bone marrow stem cell markers CD106 were negative. There was no statistical difference in the expression rate (P > 0.05).
CONCLUSION: Enzymatic pre-digestion of continuous tissue block is another way to culture adipose-derived stem cells in vitro, and the purity of adipose-derived stem cells is slightly higher than that of enzymatic digestion. The initial concentration is low and the growth cycle is slightly longer.
AIM: To investigate the growth and proliferation characteristics of adipose-derived stem cells (ADSCs) and Schwann cells (SC) from SD rats co-cultured with Transwell plate and their ability to differentiate into neural cells, and to compare the growth results of ADSCs induced by reagents (BME + BHA), so as to provide seed cells for the construction of tissue-engineered artificial nerves. The theory and basis of source.
METHODS: ADSCs were obtained from the first part of the experiment, and Schwann cells were obtained from the sciatic nerve of rats by grafting. P3 adipose-derived stem cells were divided into three groups. ADSCs and Schwann cells in group A were used to construct a co-culture system in Transwell culture plate. In group B, beta-mercaptoethanol (BME) and butyrate hydroxyanisole (BHA) were used as inducing factors of ADSCs. Cell morphology, growth of adipose-derived stem cells, axon length, expression of NF-M and GFAP were observed and analyzed statistically.
Results: Some cells of ADSCs in group A and group B differentiated into elongated processes, the growth and metabolism of ADSCs were vigorous, GFAP immunofluorescence staining was negative, and NF-M immunofluorescence staining was positive.
CONCLUSION: Co-culture of Schwann cells and ADSCs can promote the differentiation of ADSCs toward neural cells. The induction effect is similar to that induced by BME+BHA reagent, and can effectively prevent the injury and death of ADSCs. It suggests that co-culture of Schwann cells and ADSCs can be used as a method to induce ADSCs toward neural cells in the field of tissue engineering. Make use of.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2008
【分類號(hào)】：R329

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