體外誘導(dǎo)人脂肪組織來源的干細(xì)胞向角膜上皮樣細(xì)胞分化的初步研究
發(fā)布時(shí)間：2019-04-21 16:36
【摘要】：目的: 1.分離、培養(yǎng)和鑒定不同供體性狀(手術(shù)切除脂肪組織塊及吸脂術(shù)脂肪懸液)的人脂肪組織來源的干細(xì)胞(ADSCs)。觀察其生物學(xué)特性、干細(xì)胞活性的差異。 2.體外模擬角膜上皮生長(zhǎng)、分化的微環(huán)境,對(duì)不同方法提取的人ADSCs進(jìn)行單獨(dú)/聯(lián)合誘導(dǎo),探討其向角膜上皮樣細(xì)胞分化的潛在能力,及誘導(dǎo)分化的可行途徑。 方法: 1.改良方法分離、純化人ADSCs,使用改良的體外擴(kuò)增培養(yǎng)液進(jìn)行體外培養(yǎng)。 2.MTT法測(cè)定ADSCs增殖曲線。流式細(xì)胞儀檢測(cè)傳3～5代的CD29、CD34、CD49d、CD105、CD106表達(dá)率。進(jìn)行成脂、成骨多向誘導(dǎo),2周后用油紅O及堿性磷酸酶法鑒定分化能力。對(duì)2種性狀提取之細(xì)胞的表型及增殖、分化能力進(jìn)行比較。 3.應(yīng)用CFDA SE標(biāo)記人ADSCs。采用組織塊培養(yǎng)法體外提取大鼠角膜上皮細(xì)胞,并制作無上皮、內(nèi)皮細(xì)胞的大鼠角膜基質(zhì),用于ADSCs的聯(lián)合誘導(dǎo)。 4.CFDA SE標(biāo)記的人ADSCs與大鼠角膜上皮細(xì)胞各自爬片,并列培養(yǎng)于同一體系,觀察人ADSCs遷徙情況。 5.在不同培養(yǎng)液體系(角質(zhì)細(xì)胞培養(yǎng)體系、間充質(zhì)細(xì)胞體外擴(kuò)充培養(yǎng)體系及上述二者等積混合的中間體系)內(nèi)添加梯度濃度0～50ng/mL EGF,及50ng/mL EGF基礎(chǔ)上添加的10,20ng/mLbFGF,對(duì)ADSCs進(jìn)行單獨(dú)誘導(dǎo)。采取光鏡、電鏡、免疫組織化學(xué)染色、免疫熒光染色的手段,觀察誘導(dǎo)21d后ADSCs形態(tài)學(xué)的改變及角膜特異性蛋白AE5(CK3/CK12)的表達(dá)情況,比較不同培養(yǎng)體系及不同濃度生長(zhǎng)因子對(duì)ADSCs誘導(dǎo)作用的差異。 6.在Transwell體系上層放置大鼠角膜上皮細(xì)胞或角膜基質(zhì),下層放置人ADSCs,選擇不添加/添加40ng/mL EGF的角質(zhì)細(xì)胞培養(yǎng)體系對(duì)人ADSCs進(jìn)行聯(lián)合誘導(dǎo),采用免疫熒光染色法觀察誘導(dǎo)30d后ADSCs細(xì)胞角蛋白譜AE1和角膜特異性蛋白AE5的表達(dá)情況,比較添加/不添加EGF、單獨(dú)誘導(dǎo)/角膜上皮細(xì)胞聯(lián)合誘導(dǎo)/角膜基質(zhì)聯(lián)合誘導(dǎo)對(duì)人ADSCs誘導(dǎo)成功率的差異。 7.單獨(dú)誘導(dǎo)2種性狀提取的ADSCs,比較誘導(dǎo)后30d二者AE1、AE5陽性率差異。 結(jié)果: 1.不論脂肪塊還是脂肪懸液,均可通過上述方法獲取大量高純度的ADSCs。但后者的操作流程更為省時(shí)、省力,獲取的細(xì)胞量相對(duì)較多。流式細(xì)胞儀測(cè)定傳3代至傳5代的ADSCs,兩個(gè)不同供體性狀的細(xì)胞均為CD34~-、CD106~-、CD29~+、CD49d~+、CD105~+。皮下組織塊來源的ADSCs傳3代的表型CD29~+、CD49d~+細(xì)胞分別占82.5%和8.0%,傳至5代逐漸增加為89.4%、70.1%;CD105~+細(xì)胞則穩(wěn)定于80%左右。吸脂術(shù)提取的ADSCs傳3～5代的表型較為穩(wěn)定,CD29~+、CD49d~+、CD105~+表達(dá)分別在99%、40%、80%左右,與皮下組織塊組比較,傳3～5代的CD29和傳5代的CD49d陽性率有統(tǒng)計(jì)學(xué)差異(P＜0.01)。MTT比色法顯示,ADSCs增殖活性隨傳代次數(shù)增加而增加。另外,ADSCs分別進(jìn)行成脂/成骨體外誘導(dǎo),2周后油紅O、堿性磷酸酶染色,均見陽性結(jié)果,皮下組織塊組2者陽性率分別為81.6%、26.5%,吸脂術(shù)組為64.6%、29.3%。 2.CFDA SE標(biāo)記的ADSCs在營養(yǎng)狀態(tài)欠佳的角膜上皮細(xì)胞誘導(dǎo)下遷徙、并逐漸包繞、替代后者。另外,角質(zhì)細(xì)胞培養(yǎng)體系培養(yǎng)下的ADSCs向角膜上皮樣細(xì)胞分化的陽性率具有EGF濃度依賴性;bFGF則對(duì)分化有抑制性作用。而另2個(gè)體系對(duì)各濃度EGF、bFGF對(duì)ADSCs誘導(dǎo)作用均為陰性。 3.相同培養(yǎng)體系作用下的不同誘導(dǎo)組,以添加EGF刺激下的角膜基質(zhì)聯(lián)合誘導(dǎo)組AE5~+ ADSCs的比例最高,可達(dá)97.7%,AE1表達(dá)則為0,提示向成熟的角膜上皮樣細(xì)胞分化。其次為單獨(dú)誘導(dǎo)組,AE5~+比率為75.4%(添加EGF)、46.0%(未添加EGF),AE1~+比率為86.0%(添加EGF)、96.5%(未添加EGF),分化欠成熟。未添加EGF刺激下的角膜基質(zhì)聯(lián)合誘導(dǎo)組AE5、AE1陽性率最低,分別為2.4%、51.2%。 4.角質(zhì)細(xì)胞培養(yǎng)體系下,皮下組織塊組在EGF的刺激下AE5~+100%、AE1~-0%,而無EGF的作用下則分別為16.6%、97.7%。該組細(xì)胞分化能力強(qiáng)于吸脂術(shù)組。 結(jié)論: 1.本研究通過改良的方法,成功地從人皮下脂肪組織及吸脂術(shù)廢棄液中分離、培養(yǎng)了高純度脂肪組織來源的干細(xì)胞,并經(jīng)體外條件誘導(dǎo)證實(shí)具備多向分化潛能,可以作為一種優(yōu)良的組織工程、細(xì)胞治療和基因治療的種子細(xì)胞。 2.本研究中,在Transwell體系內(nèi),添加40ng/mL EGF的角質(zhì)細(xì)胞培養(yǎng)液聯(lián)合角膜基質(zhì)進(jìn)行誘導(dǎo),是體外誘導(dǎo)ADSCs向角膜上皮樣細(xì)胞分化的最佳方法。另外,皮下脂肪組織來源的ADSCs具備更好的分化能力,更適用于將來的臨床治療;而吸脂術(shù)提取的細(xì)胞由于工序相對(duì)簡(jiǎn)單、來源更為充足,更適用于實(shí)驗(yàn)室研究。 3.CFDA SE是一種穩(wěn)定的細(xì)胞示蹤劑,可作為今后體內(nèi)動(dòng)物實(shí)驗(yàn)的細(xì)胞示蹤的很好手段。
[Abstract]:Purpose: 1. Isolation, culture and identification of stem cells (ADSCs) from human adipose tissue sources of different donor properties (surgical resection of the adipose tissue mass and liposuction fat suspension) ). Observation of its biological characteristics, the difference in the activity of stem cells 2. In vitro simulation of the microenvironment of the growth and differentiation of the corneal epithelium, the ADSCs extracted from different methods were individually/ jointly induced to explore the potential for the differentiation of the corneal epithelial-like cells and the ability to induce differentiation. line approach Methods:1. The improved method was used to separate and purify human ADSCs and to use modified in vitro amplification culture. in-vitro culture of liquid.2. MTT method ADSCs proliferation curve. Flow cytometry was used to detect CD29, CD34, CD49d and CD105 of 3 to 5 generations. And the expression rate of the CD106 is higher than the expression rate of the CD106. Identification and differentiation of two traits: the phenotype and proliferation of the cells extracted from the two traits 3. Application of CFD A SE-labeled human ADSCs was used to extract the rat corneal epithelial cells in vitro by a tissue block culture method, and a rat corneal stroma with no epithelium and endothelial cells was prepared. In combination with ADSCs.4. The human ADSCs labeled by the CFDA SE and the rat corneal epithelial cells were each crawled and cultured in the same system. 5. The migration of ADSCs was observed.5. The gradient concentration of 0-50 ng/ mL of EGF and the addition of 10,20 ng/ mL of EGF on the basis of 50 ng/ mL of EGF were added in different culture solution systems (the in-vitro expansion culture system of the cell culture system and the mesenchymal cells and the above-mentioned two-product mixed intermediate system). The changes of the morphology of ADSCs and the expression of the corneal-specific protein AE5 (CK3/ CK12) were observed by light microscopy, electron microscopy, immunohistochemical staining and immunofluorescent staining. The growth of ADSCs and the expression of the corneal-specific protein AE5 (CK3/ CK12) were observed. The difference in the effect of the factor on ADSCs.6. The rat corneal epithelial cells or the corneal stroma were placed on the upper layer of the Transwell system, and the lower layer of the ADSCs was placed, and the keratinocytes were selected not to add/ add 40 ng/ mL of EGF. The expression of the human ADSCs was induced by the culture system, and the expression of the keratin-spectrum AE1 and the corneal-specific protein AE5 in the ADSCs after the induction of 30 d was observed by the immunofluorescence staining. The difference of the induction rate of human ADSCs was induced.7. The ADSCs of 2 traits were induced separately, and the results were compared. After guide 3 The positive rate of AE1 and AE5 in both AE1 and AE5 was 1. The results were as follows:1. No matter whether the fat mass or the fat suspension A large number of high-purity ADSCs can be obtained by the above method, but the latter The operation process is more time-saving and labor-saving, and the amount of the obtained cells is relatively high. The flow cytometry is used to determine the ADSCs of 3 to 5 generations, and the cells of the two different donor characters are CD34 ~-, CD106 ~-- CD29 ~ +, CD49d ~ +, CD105 ~ +, CD29 ~ +, CD49d ~ + cells were 82.5% and 8.0%, respectively, and the number of CD29 ~ +, CD49d ~ + and CD49d ~ +, CD105 ~ +. The expression of CD29 ~ +, CD49d ~ +, CD105 ~ + in CD29 ~ +, CD49d ~ + and CD105 ~ + was 99%,40% and 80%, respectively. There was a significant difference in the positive rate of CD49d (P <0.01). MTT colorimetric method The results showed that the proliferation of ADSCs increased with the increase of the number of passages. In addition, ADSCs were induced respectively in vitro and in vitro. The positive results were found in 2 weeks after the staining of oil red O and alkaline phosphatase. The positive rate of the two groups in the subcutis was 81.6%, respectively. 26.5%, in the liposuction group 64.6%, 29.3%.2. The ADSCs marked by the CFDA SE were in the vegetative state. In addition, the positive rate of ADSCs in the culture of the cell culture system to the corneal epithelial-like cells has an EGF concentration-dependent manner; bFGF has an inhibitory effect on differentiation; and the other two systems are The effect of EGF and bFGF on the induction of ADSCs was negative.3. Different induction groups under the same culture system showed the highest proportion of AE5 ~ + ADSCs with the addition of EGF. The expression of AE1 in 7% and AE1 was 0, and it was suggested to differentiate into mature corneal epithelial-like cells. Second, the rate of AE5 ~ + was 75.4% (addition of EGF), 46.0% (no EGF) and the ratio of AE1 ~ + was 86.0%. (addition of EGF), 96.5% (EGF not added), differentiation under-mature, and combination induction of corneal stroma without EGF stimulation The positive rates of AE5 and AE1 in group AE5 and AE1 were 2.4% and 51.2% respectively. -0%, and no EGF are Conclusion:1. The cell differentiation ability of the group is stronger than that of the liposuction group. Conclusion:1. The present study successfully separates the human subcutaneous fat tissue and the liposuction waste liquid by an improved method, and the stem cells derived from high-purity adipose tissue are cultured. And can be proved to be multi-directional by in-vitro condition induction. The differentiation potential can be used as a good tissue engineering, cell therapy and gene therapy seed cell. In this study, a 40 ng/ mL EGF-containing cutin cell culture solution was added to the Transwell system. It is the best way to induce the differentiation of ADSCs to the corneal epithelial-like cells in vitro. In addition, the ADSCs derived from the subcutaneous fat tissue have better differentiation and are more suitable for future clinical treatment. and the cells extracted by liposuction are relatively simple in working procedures and are more abundant in sources and are more suitable for laboratory studies.3. CF
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329
本文編號(hào)：2462371
[Abstract]:Purpose: 1. Isolation, culture and identification of stem cells (ADSCs) from human adipose tissue sources of different donor properties (surgical resection of the adipose tissue mass and liposuction fat suspension) ). Observation of its biological characteristics, the difference in the activity of stem cells 2. In vitro simulation of the microenvironment of the growth and differentiation of the corneal epithelium, the ADSCs extracted from different methods were individually/ jointly induced to explore the potential for the differentiation of the corneal epithelial-like cells and the ability to induce differentiation. line approach Methods:1. The improved method was used to separate and purify human ADSCs and to use modified in vitro amplification culture. in-vitro culture of liquid.2. MTT method ADSCs proliferation curve. Flow cytometry was used to detect CD29, CD34, CD49d and CD105 of 3 to 5 generations. And the expression rate of the CD106 is higher than the expression rate of the CD106. Identification and differentiation of two traits: the phenotype and proliferation of the cells extracted from the two traits 3. Application of CFD A SE-labeled human ADSCs was used to extract the rat corneal epithelial cells in vitro by a tissue block culture method, and a rat corneal stroma with no epithelium and endothelial cells was prepared. In combination with ADSCs.4. The human ADSCs labeled by the CFDA SE and the rat corneal epithelial cells were each crawled and cultured in the same system. 5. The migration of ADSCs was observed.5. The gradient concentration of 0-50 ng/ mL of EGF and the addition of 10,20 ng/ mL of EGF on the basis of 50 ng/ mL of EGF were added in different culture solution systems (the in-vitro expansion culture system of the cell culture system and the mesenchymal cells and the above-mentioned two-product mixed intermediate system). The changes of the morphology of ADSCs and the expression of the corneal-specific protein AE5 (CK3/ CK12) were observed by light microscopy, electron microscopy, immunohistochemical staining and immunofluorescent staining. The growth of ADSCs and the expression of the corneal-specific protein AE5 (CK3/ CK12) were observed. The difference in the effect of the factor on ADSCs.6. The rat corneal epithelial cells or the corneal stroma were placed on the upper layer of the Transwell system, and the lower layer of the ADSCs was placed, and the keratinocytes were selected not to add/ add 40 ng/ mL of EGF. The expression of the human ADSCs was induced by the culture system, and the expression of the keratin-spectrum AE1 and the corneal-specific protein AE5 in the ADSCs after the induction of 30 d was observed by the immunofluorescence staining. The difference of the induction rate of human ADSCs was induced.7. The ADSCs of 2 traits were induced separately, and the results were compared. After guide 3 The positive rate of AE1 and AE5 in both AE1 and AE5 was 1. The results were as follows:1. No matter whether the fat mass or the fat suspension A large number of high-purity ADSCs can be obtained by the above method, but the latter The operation process is more time-saving and labor-saving, and the amount of the obtained cells is relatively high. The flow cytometry is used to determine the ADSCs of 3 to 5 generations, and the cells of the two different donor characters are CD34 ~-, CD106 ~-- CD29 ~ +, CD49d ~ +, CD105 ~ +, CD29 ~ +, CD49d ~ + cells were 82.5% and 8.0%, respectively, and the number of CD29 ~ +, CD49d ~ + and CD49d ~ +, CD105 ~ +. The expression of CD29 ~ +, CD49d ~ +, CD105 ~ + in CD29 ~ +, CD49d ~ + and CD105 ~ + was 99%,40% and 80%, respectively. There was a significant difference in the positive rate of CD49d (P <0.01). MTT colorimetric method The results showed that the proliferation of ADSCs increased with the increase of the number of passages. In addition, ADSCs were induced respectively in vitro and in vitro. The positive results were found in 2 weeks after the staining of oil red O and alkaline phosphatase. The positive rate of the two groups in the subcutis was 81.6%, respectively. 26.5%, in the liposuction group 64.6%, 29.3%.2. The ADSCs marked by the CFDA SE were in the vegetative state. In addition, the positive rate of ADSCs in the culture of the cell culture system to the corneal epithelial-like cells has an EGF concentration-dependent manner; bFGF has an inhibitory effect on differentiation; and the other two systems are The effect of EGF and bFGF on the induction of ADSCs was negative.3. Different induction groups under the same culture system showed the highest proportion of AE5 ~ + ADSCs with the addition of EGF. The expression of AE1 in 7% and AE1 was 0, and it was suggested to differentiate into mature corneal epithelial-like cells. Second, the rate of AE5 ~ + was 75.4% (addition of EGF), 46.0% (no EGF) and the ratio of AE1 ~ + was 86.0%. (addition of EGF), 96.5% (EGF not added), differentiation under-mature, and combination induction of corneal stroma without EGF stimulation The positive rates of AE5 and AE1 in group AE5 and AE1 were 2.4% and 51.2% respectively. -0%, and no EGF are Conclusion:1. The cell differentiation ability of the group is stronger than that of the liposuction group. Conclusion:1. The present study successfully separates the human subcutaneous fat tissue and the liposuction waste liquid by an improved method, and the stem cells derived from high-purity adipose tissue are cultured. And can be proved to be multi-directional by in-vitro condition induction. The differentiation potential can be used as a good tissue engineering, cell therapy and gene therapy seed cell. In this study, a 40 ng/ mL EGF-containing cutin cell culture solution was added to the Transwell system. It is the best way to induce the differentiation of ADSCs to the corneal epithelial-like cells in vitro. In addition, the ADSCs derived from the subcutaneous fat tissue have better differentiation and are more suitable for future clinical treatment. and the cells extracted by liposuction are relatively simple in working procedures and are more abundant in sources and are more suitable for laboratory studies.3. CF
【學(xué)位授予單位】：中國協(xié)和醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R329
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