同种异体骨髓间充质干细胞穴位移植1天与7天对急性心肌梗死后心肌细胞凋亡的对比研究

Homogeneous Foreign Body Marrow Mesenchyma Stem Cell Acupuncture Point Transplant One Day with Seven Day of the Acute Heart Stem Back of the Body Muscle Cell Perishes Weakly Contrast Research

目的:进行兔骨髓间充质干细胞(MSCs)穴位移植1天与7天对梗死区心肌细胞凋亡的对比研究。方法:将已建立心肌梗死模型的日本大耳白兔随机分为3组,包括模型组、穴位移植干细胞1天组、穴位移植干细胞7天组。用开胸结扎冠状动脉前降支的方法建立急性心肌梗死模型,将梗死后的兔分两组分别在1天与7天穴位注射相同数量的已经过分离、培养、扩增、标记后的MSCs悬液。采用脱氧核苷酸末端转移酶介导原位缺口标记(TUNEL法)确定细胞凋亡数,免疫组化SABC方法及RT-PCR和蛋白印迹法观察心肌细胞中Fas、FasL、Bcl-2蛋白及mRNA的表达。结果:(1)免疫组化示:梗死区凋亡细胞的表达,模型组高于正常组、穴位注射干细胞1天组和穴位注射干细胞7天组(P<0.05),穴位注射干细胞7天组低于穴位注射干细胞1天组(但无统计学差异P>0.05);Fas蛋白的表达,模型组高于正常组、穴位注射干细胞1天组和穴位注射干细胞7天组,穴位注射干细胞7天组低于穴位注射干细胞1天组(P<0.05);FasL蛋白的表达,穴位注射干细胞1天组高于正常组、模型组、穴位注射干细胞7天组(P<0.05);Bcl-2蛋白的表达,模型组低于正常组、穴位注射干细胞1天组和穴位注射干细胞7天组(P<0.05),穴位注射干细胞7天组高于穴位注射干细胞1天组(但无统计学差异P>0.05)。(2)RT-PCR与Western Blotting均显示:Fas、FasL、Bcl-2蛋白mRNA的表达穴位移植干细胞1天均高于穴位移植干细胞7天组。结论:(1)穴位移植骨髓间充质干细胞可使梗死及周围区心肌细胞凋亡数减少,其机制可能是通过Fas、FasL蛋白的减少、Bcl-2蛋白的增多来调节的。(2)穴位移植骨髓间充质干细胞7天较穴位移植骨髓间充质干细胞1天更能减少凋亡细胞的数量。

Objective： To carry out the contrast research of apoptotic cardiomyocytes between the rabbit marrow mesenehymal stem cells（MSCs） acupuncture point transplantation for one day and for seven days. Methods ：The Japanese big ear white rabbits which have been set up as the myocardial infarction model were randomly divided into 3 groups, including the model group, the group of acupuncture point transplant stem ceils for one day, and the group of acupuncture point transplant stem ceils for seven day. Thoracotomy with anterior descending coronary artery ligation method is used to set up acute myocardial infarction medel. The rabbits will be divided into two groups after infarction, and respectively ,in the acu- point of one day and seven days, will be injected in the same volume of MSCs hanging liquid which has been isolated, cultured, amplified and marked. Using terminal deoxynucleotide transferase-mediated in situ nick markers（TUNEL） to determine the number of apoptotic, and immunohistechemistry SABC methods and RT - PCR and Western blot to observe the expression of Fas, FasL, Bcl -2 protein and mRNA in myocardial cells. Results：（ 1 ）Immunohistochemistry showing：Concerned with the expression of apoptotic cells in the infarcted area,the model group is higher than the normal group, the group with acupoint injected stem cells for one day and the group with acupoint injected stem cells for seven days （ P 〈 0. 05 ） ,the group with acupoint injected stem cens for seven days is lower than the group with acupoint injected stem cells for one day （ but non-statistics difference P 〉 0. 05） ;concerned with Fas protein expression, the model group is higher than the normal group,the group with acupoint injected stem cells for one day and the group with acupoint injected stem cells for seven days, the group with acupoint injected stem cells for seven days is lower than the group with acupoint injected stem cells for one day（P 〈 0. 05 ） ; concerned with the expression of FasL protein, the group with acupoint injected stem cells for one day is higher than the normal group, the model group, and the group with acupoint injected stem cells for seven days （P 〈 0. 05 ） ;concerned with the expression of Bcl -2 protein, the model group is lower than the normal group, the group with acupoint injected stem cells for one day and the group with acupoint injected stem cells for seven days （ P 〈 0. 05 ）, the group with acupoint injected stem cells for seven days is higher than the group with acupoint injected stem ceils for one day（ but non-statistics difference P 〉 0.05 ）. （2） RT - PCR and Western Blotting showing ： Concerned with the expression of Fas, FasL, Bcl - 2 protein, mRNA, the group with acupoint transplanted stem cells for one day is higher than the group with acupoint transplanted stem cells for seven days. Conclusion ： （ 1 ） The acupuncture point transplanted with marrow mesenchymal stem cells enables to reduce the number of apoptotic cardiomyocytes in the infarcted and the surrounding area. Its mechanism is possibly that it is adjusted by the reduction of Fas, FasL protein, and the increase of Bcl- 2 protein. （2）Transplanting marrow mesenchymal stem cells in acupuncture point for seven days is able to reduce more apoptotic cells compared with transplanting marrow mcsenchyma stem cells in acupuncture point for one day.