【摘要】：目的：觀察大鼠骨髓間充質(zhì)干細(xì)胞(marrow mesenchymal stem cells, MSCs)與竇房結(jié)(sinoatrial node, SAN)組織塊混合誘導(dǎo)培養(yǎng)后Cx40及HCN4的表達(dá)情況,探討大鼠骨髓間充質(zhì)干細(xì)胞向竇房結(jié)細(xì)胞分化的可能性。方法：(1)大鼠骨髓間充質(zhì)干細(xì)胞的取材：將健康SD大鼠用75%酒精直接浸泡處死,無菌條件下解剖出股骨、脛骨。用PBS液沖洗股骨、脛骨骨髓腔,離心管收集富含骨髓間充質(zhì)干細(xì)胞的細(xì)胞懸液。(2)大鼠骨髓間充質(zhì)干細(xì)胞的分離、培養(yǎng)：將富含骨髓間充質(zhì)干細(xì)胞的細(xì)胞懸液離心,吸去上清液,加入含10%胎牛血清的DMEM高糖培養(yǎng)液制成細(xì)胞懸液,接種于細(xì)胞培養(yǎng)瓶。采用貼壁篩選法分離、純化大鼠骨髓間充質(zhì)干細(xì)胞,置于37℃含5%CO2的恒溫孵箱中培養(yǎng),待細(xì)胞融合接近80%時(shí)用0.25%的胰蛋白酶消化、傳代。(3)MSCs的純度及活性鑒定：采用細(xì)胞免疫熒光法,利用CD34、CD44抗體鑒定傳至第3代的大鼠骨髓間充質(zhì)干細(xì)胞純度,同時(shí)用臺(tái)盼藍(lán)染色鑒定第3代大鼠骨髓間充質(zhì)干細(xì)胞的活性。(4)接種MSCs:取經(jīng)過純度和活性鑒定的第3代MSCs細(xì)胞,用熒光染料CFSE(5, 6-carboxyfluorescein diacetate, N-succinimidyl ester)標(biāo)記,以確保實(shí)驗(yàn)的細(xì)胞是來源于第3代MSCs,然后以相同的細(xì)胞濃度(1×105/ml)分別接種于六孔培養(yǎng)板的培養(yǎng)孔內(nèi),或者培養(yǎng)孔內(nèi)的蓋玻片上制成細(xì)胞爬片。(5)大鼠竇房結(jié)組織的取材：用3%戊巴比妥鈉腹腔注射麻醉，，無菌條件下取出SD大鼠心臟,PBS液反復(fù)沖洗。于右心房竇房結(jié)區(qū)域選取竇房結(jié)組織,剪成約0.3cm×0.3cm大小的組織塊,立即置入4℃無菌的PBS液中。(6)混合培養(yǎng)：將組織塊分別置入培養(yǎng)孔的邊緣與大鼠MSCs混合培養(yǎng),但不與MSCs直接接觸。(7)分組：對照組(A組)：單獨(dú)培養(yǎng)大鼠MSCs1周；實(shí)驗(yàn)組：將大鼠MSCs與竇房結(jié)組織塊混合培養(yǎng),分別誘導(dǎo)培養(yǎng)1周(B組)、2周(C組)和3周(D組)。(8)篩選細(xì)胞：培養(yǎng)結(jié)束后,于熒光顯微鏡下篩選出各組中CFSE標(biāo)記率較高(90%)且分布均勻的細(xì)胞,以保證被用來檢測的細(xì)胞系來源于第3代MSCs,分別做Cx40和HCN4的檢測。(9)細(xì)胞Cx40及HCN4的MIOD檢測：75%酒精固定細(xì)胞爬片,采用免疫組織化學(xué)的方法(SP法)染色,運(yùn)用Image-pro plus 5.0圖像分析軟件鑒定各組細(xì)胞表達(dá)Cx40和HCN4的平均積分光密度值(mean integrated optical density, MIOD)值。(10)細(xì)胞Cx40及HCN4的mRNA檢測：采用實(shí)時(shí)熒光定量PCR (Real-time fluorescent quantitative PCR, RT-PCR)的方法,檢測各組細(xì)胞中Cx40、HCN4的mRNA表達(dá)水平。(11)統(tǒng)計(jì)分析：實(shí)驗(yàn)數(shù)據(jù)采用SPSS13.0軟件進(jìn)行統(tǒng)計(jì)分析,組間比較采用方差分析的方法。結(jié)果：(1)采用細(xì)胞免疫熒光法鑒定實(shí)驗(yàn)分離、培養(yǎng)、純化的第3代MSCs,在每熒光顯微鏡視野下MSCs占80%以上；同時(shí)用臺(tái)盼藍(lán)染色判定MSCs的活性在90%以上。(2)實(shí)驗(yàn)組細(xì)胞Cx40、HCN4的MIOD值都比對照組高,即實(shí)驗(yàn)組細(xì)胞Cx40、HCN4的表達(dá)比對照組強(qiáng),差異有統(tǒng)計(jì)學(xué)意義(p＜0.01)；且隨著混合培養(yǎng)時(shí)間的延長,Cx40及HCN4的表達(dá)逐漸增加,實(shí)驗(yàn)組間差異均有統(tǒng)計(jì)學(xué)意義(p0.05)。(3)實(shí)驗(yàn)組細(xì)胞Cx40、HCN4的mRNA表達(dá)水平都比對照組高,差異有統(tǒng)計(jì)學(xué)意義(p＜0.01)；并且培養(yǎng)時(shí)間越長,Cx40及HCN4的mRNA表達(dá)越明顯,組間差異均有統(tǒng)計(jì)學(xué)意義(p0.05)。結(jié)論：(1)本實(shí)驗(yàn)采用的大鼠MSCs的取材、以及貼壁法分離、培養(yǎng)細(xì)胞的方法可靠,細(xì)胞傳至第3代后能獲得活性和純度均較高的MSCs。(2)將大鼠MSCs與竇房結(jié)組織塊體外混合培養(yǎng),誘導(dǎo)后細(xì)胞Cx40及HCN4高表達(dá)。(3)大鼠MSCs經(jīng)過與竇房結(jié)組織塊體外混合培養(yǎng)后,具有向竇房結(jié)細(xì)胞分化的可能。
[Abstract]:Objective: To study the expression of Cx40 and HCN4 in rat bone marrow-derived mesenchymal stem cells (MSCs), and to explore the possibility of the differentiation of bone marrow-derived mesenchymal stem cells into the cells of the rat. Methods: (1) The rat bone marrow mesenchymal stem cells were obtained: the healthy SD rats were directly soaked and killed with 75% alcohol, and the femur and the tibia were dissected under sterile conditions. The femur, the tibial bone marrow cavity, and the centrifuge tube were washed with PBS to collect the cell suspension rich in the bone marrow mesenchymal stem cells. (2) separating and culturing the rat bone marrow mesenchymal stem cells, centrifuging the cell suspension rich in the bone marrow mesenchymal stem cells, sucking the supernatant, adding the DMEM high-glucose culture solution containing 10% fetal bovine serum to prepare the cell suspension, and inoculating the cell culture bottle. The bone marrow-derived mesenchymal stem cells were isolated and purified in a constant-temperature incubator containing 5% CO2 at 37 鈩

本文編號(hào)：2442389