携带OPG基因的慢病毒转染骨髓间充质干细胞治疗骨质疏松大鼠的效果研究

发布时间：2018-07-07 21:59

本文选题：骨保护素 + 骨髓间充质干细胞　；参考：《山西医科大学》2015年硕士论文

【摘要】：目的:将携带有OPG基因的慢病毒体外转染至大鼠BMSCs,并检测其表达结果,为体内基因治疗骨质疏松提供种子细胞。然后将转染成功的BMSCs注射到骨质疏松大鼠的骨髓腔内,分不同时段测定相关指标,观察测定其治疗效果,探索其治疗骨质疏松症的可行性。方法:体外实验选取清洁级2月龄的雌性SD大鼠2只,无菌条件下取其股骨和胫骨,分离和培养BMSCs,第三代时进行慢病毒转染,设立对照组(未经任何特殊处理)、未携带OPG基因慢病毒载体组(转染PGC-FU-RFP)、携带OPG基因慢病毒载体组(转染PGC-FU-OPG-RFP)。72h后,免疫荧光显微镜观察其荧光表达,流式细胞仪测定表达效率。体内试验选取健康6月龄雌性SD大鼠30只,随机分成5组,每组各6只:A组:假手术组;B组:双侧卵巢切除组。造模三个月后测定大鼠的骨密度。造模成功后,给予各组左股骨髓腔注射:B1组生理盐水;B2组BMSCs;B3组未转染OPG基因的BMSCs;B4组转染OPG基因的BMSCs。分别于注射后第6、12周后测定大鼠的骨密度,血清钙、磷浓度、血清骨碱性磷酸酶(B-ALP)及I型胶原交联C-末端肽(CTX-1),X线摄片观察股骨大体情况。12周后颈椎脱臼处死大鼠,进行双侧股骨标本大体观察,右侧股骨三点弯曲试验,并进行统计学分析。结果:体外实验成功培养出第三代大鼠的BMSCs,以MOI=100转染,72h后荧光表达满意,转染效率达70—80%。体内试验,造模前:骨密度测定,各组间无统计学差异。造模3月后:骨密度、血磷B组明显低于A组;血钙、B-ALP、CTX-1 B组明显高于A组,且有统计学意义(P0.05)。骨髓腔注射BMSCs或生理盐水6周后:骨密度B组仍低于A组,B4组高于其余B组,B2、B3组均高于B1组;血钙B4组低于其余B组,血磷B4组高于其余B组,明显低于A组;B-ALP和CTX-1,B组仍明显高于A组,B4组明显低于其余B组,且有统计学意义(P0.05)。骨髓腔注射BMSCs或生理盐水12周后,除血磷、B-ALP、CTX-1 B4组与A组无差异外,其余比较结果与6周后相同,且有统计学意义(P0.05)。骨髓腔注射BMSCs或生理盐水12周后,B4组骨密度明显高于6周后,B-ALP、CTX-1明显低于6周后,且有统计学意义(P0.05),血钙、血磷的变化不明显。不同时期各组大鼠的股骨X线片比较,变化不明显。各组大鼠股骨标本差异不明显。股骨三点弯曲试验示:右侧股骨最大载荷、最大挠度、最大能量吸收、最大应力及弹性模量,B4组略低于A组,明显高于其余B组,且有统计学意义(P0.05)。结论:携带OPG基因的慢病毒能有效转染BMSCs,并能稳定地表达,当MOI=100时,转染效率可达70-80%。将携带OPG基因的BMSCs对骨质疏松大鼠有以下效果:(1)可以有效改善骨质疏松大鼠的骨密度继续下降的趋势;(2)可以有效抑制破骨细胞活性,降低骨质疏松风险,提高抗骨折能力。
[Abstract]:Aim: to transfect lentivirus carrying OPG gene into BMSCs of rats in vitro and detect its expression results to provide seed cells for gene therapy of osteoporosis in vivo. Then the successful BMSCs were injected into the medullary cavity of osteoporosis rats, and the related indexes were measured in different periods, the therapeutic effect was observed and the feasibility of treating osteoporosis was explored. Methods: two female Sprague-Dawley rats of 2 months of clean grade were selected in vitro. Femur and tibia were isolated and cultured in aseptic condition. The third generation of BMSCs was transfected with lentivirus. In the control group (without any special treatment), the lentivirus vector group did not carry the OPG gene (PGC-FU-RFP) and the lentivirus vector carrying the OPG gene (PGC-FU-OPG-RFP) for 72 hours. The fluorescence expression was observed by immunofluorescence microscope and the expression efficiency was measured by flow cytometry. In vivo test, 30 healthy 6-month-old female SD rats were randomly divided into 5 groups, 6 in each group: group A: sham operation group: group B: bilateral ovariectomized group. Bone mineral density (BMD) was measured three months later. After the model was successfully established, each group was injected into the left femoral medullary cavity with normal saline group B _ 1 and B _ 2 group B _ (2) with BMSCs / B _ (3) group without OPG gene transfection and BMSCs / B _ (4) group with OPG gene transfection. Bone mineral density (BMD), serum calcium and phosphorus concentrations, serum bone alkaline phosphatase (B-ALP) and type I collagen cross-linked C-terminal peptide (CTX-1) X-ray films were measured at the 6th week after injection. Gross observation of bilateral femur specimens and three-point bending test of right femur were carried out. Results: BMSCs of the third generation of rats were successfully cultured in vitro. After transfection with moi 100 for 72 hours, the fluorescent expression was satisfactory, and the transfection efficiency was 70-80%. In vivo test, before modeling: bone mineral density measurement, there was no statistical difference among the groups. After 3 months of modeling, BMD in group B was significantly lower than that in group A, and CTX-1 in group B was significantly higher than that in group A (P0.05). Six weeks after BMSCs or normal saline were injected into bone marrow cavity, BMD in group B was still lower than that in group A, and higher in group B than in group B, but higher in group B3 than in group B1, in group B, it was higher in group B than in group B. Group B was significantly lower than group A (P 0.05). Group B was still significantly higher than group A (P 0.05). Group B was significantly lower than group B (P 0.05). After 12 weeks of injection of BMSCs or normal saline into bone marrow, the results of BMSCs were the same as those of group A (P0.05), except that there was no difference between group A and group B (P 0.05). After 12 weeks of BMSCs or normal saline injection, the BMD in B4 group was significantly higher than that after 6 weeks (P0.05), and the changes of serum calcium and phosphorus were not significant. There was no obvious change in X-ray film of femur in different groups. There was no significant difference in femur specimens among the groups. Three-point bending test of femur showed that the maximum load, maximum deflection, maximum energy absorption, maximum stress and modulus of elasticity of right femur in group B 4 were slightly lower than those in group A, which were significantly higher than those in group B (P0.05). Conclusion: lentivirus carrying OPG gene can effectively transfect BMSCs and express stably. When MOI = 100, the transfection efficiency can reach 70-80. The effects of BMSCs carrying OPG gene on osteoporosis rats were as follows: (1) it could effectively improve the decreasing trend of bone mineral density in osteoporosis rats; (2) it could effectively inhibit the activity of osteoclasts, reduce the risk of osteoporosis and improve the ability of anti-fracture.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R580

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