【摘要】： 目的: 1.通過(guò)對(duì)兔脂肪干細(xì)胞的分離、培養(yǎng)及鑒定,獲得純化的兔脂肪干細(xì)胞(ADSCs);建立一種穩(wěn)定、高效的兔ADSCs的培養(yǎng)方法。 2.尋求一種BMP-9基因轉(zhuǎn)染靶細(xì)胞ADSCs的穩(wěn)定的實(shí)驗(yàn)方法;證實(shí)BMP-9基因轉(zhuǎn)染后的ADSCs可向成骨細(xì)胞分化,以獲得可用于骨組織工程的基因修飾的種子細(xì)胞。 方法: 1.ADSCs的分離、培養(yǎng)及鑒定:采用I型膠原酶消化法獲取原代ADSCs。擴(kuò)增和純化ADSCs后,顯微鏡下觀察兔ADSCs形態(tài),MTT、細(xì)胞計(jì)數(shù)分析ADSCs的體外增殖情況,免疫組化檢測(cè)ADSCs表面抗原CD29、CD44的表達(dá)。 2.BMP-9轉(zhuǎn)染ADSCs的實(shí)驗(yàn):載BMP-9的腺病毒直接感染P3代的ADSCs。熒光顯微鏡觀察轉(zhuǎn)染效果,流式細(xì)胞儀測(cè)定轉(zhuǎn)染率,RT-PCR檢測(cè)轉(zhuǎn)染后ADSCs內(nèi)BMP-9 mRNA的表達(dá)。堿性磷酸酶活性、堿性磷酸酶和茜素紅鈣結(jié)節(jié)染色檢測(cè)轉(zhuǎn)染后ADSCs的成骨活性。 結(jié)果: 1.ADSCs的分離、培養(yǎng)及鑒定:(1)分離、培養(yǎng)的原代ADSCs細(xì)胞形態(tài)呈梭形或多角狀,類似成纖維細(xì)胞,團(tuán)簇狀生長(zhǎng),在10 d左右達(dá)到90%融合。(2)經(jīng)過(guò)傳代、純化的ADSCs呈成纖維細(xì)胞樣,分布均勻、大小一致、細(xì)胞緊密排列,漩渦狀生長(zhǎng),5 d左右達(dá)90%融合,顯示出活躍的增殖能力。(3)生長(zhǎng)曲線顯示:1~3 d為傳代培養(yǎng)的滯留期,4~6 d為對(duì)數(shù)增殖期,7~9 d為生長(zhǎng)抑制期,此時(shí)細(xì)胞生長(zhǎng)速度變慢。(4)累計(jì)倍增曲線顯示:群體倍增時(shí)間大約為55 h,P3代細(xì)胞增殖速度最快,P8代以后的細(xì)胞增殖速度減慢,細(xì)胞出現(xiàn)衰老征象。(5)免疫組化染色顯示:ADSCs細(xì)胞內(nèi)表面抗原CD29、CD44均陽(yáng)性表達(dá)。 2.BMP-9轉(zhuǎn)染ADSCs的實(shí)驗(yàn):(1)BMP-9基因腺病毒載體可成功轉(zhuǎn)染ADSCs。(2)腺病毒介導(dǎo)的BMP-9基因轉(zhuǎn)染ADSCs后24 h即有熒光表達(dá),并隨時(shí)間延長(zhǎng)熒光強(qiáng)度逐漸增強(qiáng),轉(zhuǎn)染3d轉(zhuǎn)染效率達(dá)到80%以上。(3)轉(zhuǎn)染后ADSCs停滯期略延長(zhǎng),數(shù)量輕度下降,倍增時(shí)間稍延長(zhǎng),但不影響細(xì)胞增殖。(4) RT-PCR檢測(cè)結(jié)果顯示,各轉(zhuǎn)染組細(xì)胞內(nèi)hBMP-9的mRNA持續(xù)陽(yáng)性表達(dá),未轉(zhuǎn)染組未見(jiàn)陽(yáng)性條帶。(5)ADSCs轉(zhuǎn)染后,ALP染色及茜素紅鈣結(jié)節(jié)染色為陽(yáng)性,細(xì)胞內(nèi)ALP活性呈增長(zhǎng)趨勢(shì),轉(zhuǎn)染組顯著高于未轉(zhuǎn)染組。 結(jié)論: 1.從兔脂肪組織中分離、培養(yǎng)、純化的ADSCs具有明顯的干細(xì)胞特征,體外生長(zhǎng)旺盛,擴(kuò)增迅速,長(zhǎng)期傳代仍能保持穩(wěn)定的增殖能力,為BMP-9轉(zhuǎn)染ADSCs誘導(dǎo)成骨的實(shí)驗(yàn)奠定了基礎(chǔ)。 2.腺病毒介導(dǎo)的BMP-9基因可以安全、有效地轉(zhuǎn)染ADSCs,其轉(zhuǎn)染的BMP-9基因可獲得較高水平的表達(dá),且具有明顯的誘導(dǎo)ADSCs向成骨細(xì)胞轉(zhuǎn)化的作用;而攜帶人全長(zhǎng)BMP-9基因的腺病毒轉(zhuǎn)染ADSCs可作為研究BMP-9誘導(dǎo)ADSCs成骨作用實(shí)驗(yàn)的可靠方法。
[Abstract]:Objective:
1. through isolation, culture and identification of rabbit adipose derived stem cells, purified rabbit adipose derived stem cells (ADSCs) were obtained, and a stable and efficient method for ADSCs culture was established.
2. seek a stable experimental method of transfection of BMP-9 gene to target cell ADSCs, and confirm that ADSCs transfected by BMP-9 gene can differentiate into osteoblasts to obtain gene modified seed cells that can be used for bone tissue engineering.
Method:
The separation, culture and identification of 1.ADSCs: after I type collagenase digestion was used to obtain the original ADSCs. amplification and purification of ADSCs, the ADSCs morphology of rabbit was observed under microscope, MTT, cell count was used to analyze the proliferation of ADSCs in vitro, and the expression of ADSCs surface antigen CD29 and CD44 was detected by immunohistochemistry.
The transfection of ADSCs by 2.BMP-9: the adenovirus carrying BMP-9 was directly infected with the P3 generation ADSCs. fluorescence microscope to observe the transfection effect. The transfection rate was measured by flow cytometry. The expression of BMP-9 mRNA in ADSCs after the transfection was detected by RT-PCR. The activity of alkaline phosphatase, alkaline phosphatase and alizarin red calcium nodule staining were used to detect the osteogenic activity of ADSCs in the transfected ADSCs.
Result:
1.ADSCs isolation, culture and identification: (1) isolated, cultured primary ADSCs cells have spindle or multi angle shape, similar to fibroblasts, cluster like growth and 90% fusion at about 10 d. (2) after passage, the purified ADSCs is fibroblast like, evenly distributed and uniform in size, cells closely arranged, whirlpool, 5 d to 90% fusion, (3) the growth curve showed that 1~3 D was the stagnation period of subculture, 4~6 D was a logarithmic proliferation period, 7~9 D was a growth inhibition period, and the growth rate of cell slowed. (4) the cumulative doubling curve showed that the multiplication time of the population was about 55 h, the proliferation rate of P3 generation was the fastest, and the proliferation rate slowed down after the P8 generation. The cells showed signs of aging. (5) immunohistochemical staining showed that the surface antigen CD29 and CD44 of ADSCs cells were all positive.
2.BMP-9 transfection of ADSCs: (1) BMP-9 gene adenovirus vector could be transfected successfully with ADSCs. (2) adenovirus mediated BMP-9 gene transfection to ADSCs after transfection to 24 h, and the fluorescence intensity was gradually increased with time, and the transfection efficiency of 3D transfection reached more than 80%. (3) the stagnation period of ADSCs was slightly prolonged, the number of 3D was slightly decreased and the doubling time was delayed slightly after transfection. Long, but did not affect cell proliferation. (4) the results of RT-PCR detection showed that the mRNA of hBMP-9 in the cells of each transfection group was positive and no positive bands were not found in the untransfected group. (5) after ADSCs transfection, ALP staining and alizarin red calcium nodule staining were positive, and the intracellular ALP activity was increasing, and the transfection group was significantly higher than that of the untransfected group.
Conclusion:
1. isolated, cultured and purified ADSCs from rabbit adipose tissue has obvious characteristics of stem cell. It grows exuberant in vitro, expands rapidly, and can still maintain stable proliferative ability for a long time. It lays a foundation for the experiment of BMP-9 transfection of ADSCs to induce osteogenesis.
2. adenovirus mediated BMP-9 gene can be safely and effectively transfected to ADSCs. The transfected BMP-9 gene can obtain a high level of expression, and it can obviously induce the transformation of ADSCs into osteoblasts, and the transfection of ADSCs with the adenovirus carrying human BMP-9 gene can be used as a reliable method for the study of BMP-9 induced ADSCs osteogenesis.
【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】：2009
【分類號(hào)】：R329

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