骨髓间充质干细胞治疗大鼠骨关节炎的实验研究

摘要

Objective:To investigate the efficacy of single time intra-articular different concentration of allogeneic bone marrow mesenchymal stem cells ( BM-MSCs ) injection in the treatment of Sprague-Dawley ( SD) rat model of osteoarthritis ( OA) .Methods: In the study, 32 SD rats were equally ran-domized into 4 groups:control group, high concentration group (1 ×107/mL BM-MSCs), low concentra-tion group (5 ×106/mL BM-MSCs) and high vs.low concentration group.The two knees of each rat were set up to a pair.The induction of OA was performed surgically randomly at one side in model group, and bilaterally in the other groups, which were through anterior cruciate ligament transaction ( ACLT) and medial meniscus excising.After the operation, the SD rats were allowed free movement.Four weeks later, different concentrations of allogeneic BM-MSCs isolated from the SD rats, expanded in vitro and suspended in phosphate buffered solution( PBS) were delivered in the articular cavity of both knees;PBS was used as the control.After injection, we excised the femoral nerve and sciatic nerve to disuse the low limb.The cartilage histological sections of knees were scored by Mankin scoring system to assess the se-verity of the pathology.mRNA of collagen Ⅱwas detected by real time polymerase chain reaction ( RT-PCR) .eGFP was detected by fluorescence microscope.Assessments were carried out 4 weeks after the operation in model group, and 3 weeks after injection in the other groups.Results:Mankin scores of the BM-MSCs side and control side were 6.60 ±0.40 vs.10.00 ±0.32 in low concentration group ( P<0.05), and 5.40 ±0.51 vs.9.60 ±0.51 in high concentration group (P<0.05).Mankin scores of high sv.low concentration group were 6.40 ±0.51 vs.7.60 ±0.75 (P>0.05).mRNA expression of collagen Ⅱ of the BM-MSCs side in low concentration group was 106%±1%in contrast to the control side.As in high concentration group it was 108%±1%, and 102%±1%in high vs. low concentra-tion group.Labeled BM-MSCs were detected unexpectedly in the synovial membrane but not in cartilage tissue three weeks from injection.Conclusion:BM-MSCs could promote cartilage repair and inhibit OA progression through a trophic mechanism.There was no difference between the two concentrations.%目的：研究不同浓度骨髓间充质干细胞（ bone marrow mesenchymal stem cells，BM-MSCs）对骨关节炎（ oste-oarthritis，OA）的治疗效果，并探究其治疗机制。方法：8周龄近交系SD大鼠32只随机分成4组，每组8只，均采用自身双侧对照研究：对照组、高浓度组（1×107／mL BM-MSCs）、低浓度组（5×106／mL BM-MSCs）及高／低浓度对比组。应用改良Hulth方法诱导膝关节OA。对照组一侧行手术，另一侧行假手术，其余各组均行双侧手术。术后4周处死对照组大鼠采集双侧膝关节标本。各试验组大鼠膝关节内注入相应浓度的BM-MSCs或磷酸盐缓冲液（ phosphate buffered solution，PBS），切断大鼠双侧坐骨神经及股神经使下肢制动，注射3周后处死各组大鼠并收取双侧膝关节标本。应用Mankin组织学评分评价OA病变程度，RT-PCR检测软骨Ⅱ型胶原mRNA表达，荧光显微镜观察荧光蛋白标记的BM-MSCs在膝关节内分布情况。结果：高浓度组、低浓度组BM-MSCs侧软骨组织标本Mankin评分均明显低于对照侧（5．40±0．51 vs．9．60±0．51；6．60±0．40 vs．10．00±0．32；P均＜0．05），高／低浓度对比组高浓度侧Mankin评分略低于低浓度侧（6．40±0．51 vs．7．60±0．75，P＞00．5）。 RT-PCR结果显示，高浓度组、低浓度组BM-MSCs侧软骨Ⅱ型胶原mRNA含量分别为对照侧的108％±1％和106％±1％，高／低浓度对比组高浓度侧Ⅱ型胶原mRNA 含量是低浓度侧的102％±1％。荧光显微镜显示，在软骨表面未见绿色荧光蛋白表达，而在滑膜组织内可见绿色荧光表达。结论：关节腔内注入BM-MSCs可能通过间接机制对OA软骨病变起到保护作用，两种浓度治疗效果无差异。