本文選題：骨保護(hù)素 + 骨髓間充質(zhì)干細(xì)胞��； 參考：《山西醫(yī)科大學(xué)》2015年碩士論文

【摘要】：目的:將攜帶有OPG基因的慢病毒體外轉(zhuǎn)染至大鼠BMSCs,并檢測(cè)其表達(dá)結(jié)果,為體內(nèi)基因治療骨質(zhì)疏松提供種子細(xì)胞。然后將轉(zhuǎn)染成功的BMSCs注射到骨質(zhì)疏松大鼠的骨髓腔內(nèi),分不同時(shí)段測(cè)定相關(guān)指標(biāo),觀察測(cè)定其治療效果,探索其治療骨質(zhì)疏松癥的可行性。方法:體外實(shí)驗(yàn)選取清潔級(jí)2月齡的雌性SD大鼠2只,無(wú)菌條件下取其股骨和脛骨,分離和培養(yǎng)BMSCs,第三代時(shí)進(jìn)行慢病毒轉(zhuǎn)染,設(shè)立對(duì)照組(未經(jīng)任何特殊處理)、未攜帶OPG基因慢病毒載體組(轉(zhuǎn)染PGC-FU-RFP)、攜帶OPG基因慢病毒載體組(轉(zhuǎn)染PGC-FU-OPG-RFP)。72h后,免疫熒光顯微鏡觀察其熒光表達(dá),流式細(xì)胞儀測(cè)定表達(dá)效率。體內(nèi)試驗(yàn)選取健康6月齡雌性SD大鼠30只,隨機(jī)分成5組,每組各6只:A組:假手術(shù)組;B組:雙側(cè)卵巢切除組。造模三個(gè)月后測(cè)定大鼠的骨密度。造模成功后,給予各組左股骨髓腔注射:B1組生理鹽水;B2組BMSCs;B3組未轉(zhuǎn)染OPG基因的BMSCs;B4組轉(zhuǎn)染OPG基因的BMSCs。分別于注射后第6、12周后測(cè)定大鼠的骨密度,血清鈣、磷濃度、血清骨堿性磷酸酶(B-ALP)及I型膠原交聯(lián)C-末端肽(CTX-1),X線攝片觀察股骨大體情況。12周后頸椎脫臼處死大鼠,進(jìn)行雙側(cè)股骨標(biāo)本大體觀察,右側(cè)股骨三點(diǎn)彎曲試驗(yàn),并進(jìn)行統(tǒng)計(jì)學(xué)分析。結(jié)果:體外實(shí)驗(yàn)成功培養(yǎng)出第三代大鼠的BMSCs,以MOI=100轉(zhuǎn)染,72h后熒光表達(dá)滿意,轉(zhuǎn)染效率達(dá)70—80%。體內(nèi)試驗(yàn),造模前:骨密度測(cè)定,各組間無(wú)統(tǒng)計(jì)學(xué)差異。造模3月后:骨密度、血磷B組明顯低于A組;血鈣、B-ALP、CTX-1 B組明顯高于A組,且有統(tǒng)計(jì)學(xué)意義(P0.05)。骨髓腔注射BMSCs或生理鹽水6周后:骨密度B組仍低于A組,B4組高于其余B組,B2、B3組均高于B1組;血鈣B4組低于其余B組,血磷B4組高于其余B組,明顯低于A組;B-ALP和CTX-1,B組仍明顯高于A組,B4組明顯低于其余B組,且有統(tǒng)計(jì)學(xué)意義(P0.05)。骨髓腔注射BMSCs或生理鹽水12周后,除血磷、B-ALP、CTX-1 B4組與A組無(wú)差異外,其余比較結(jié)果與6周后相同,且有統(tǒng)計(jì)學(xué)意義(P0.05)。骨髓腔注射BMSCs或生理鹽水12周后,B4組骨密度明顯高于6周后,B-ALP、CTX-1明顯低于6周后,且有統(tǒng)計(jì)學(xué)意義(P0.05),血鈣、血磷的變化不明顯。不同時(shí)期各組大鼠的股骨X線片比較,變化不明顯。各組大鼠股骨標(biāo)本差異不明顯。股骨三點(diǎn)彎曲試驗(yàn)示:右側(cè)股骨最大載荷、最大撓度、最大能量吸收、最大應(yīng)力及彈性模量,B4組略低于A組,明顯高于其余B組,且有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:攜帶OPG基因的慢病毒能有效轉(zhuǎn)染BMSCs,并能穩(wěn)定地表達(dá),當(dāng)MOI=100時(shí),轉(zhuǎn)染效率可達(dá)70-80%。將攜帶OPG基因的BMSCs對(duì)骨質(zhì)疏松大鼠有以下效果:(1)可以有效改善骨質(zhì)疏松大鼠的骨密度繼續(xù)下降的趨勢(shì);(2)可以有效抑制破骨細(xì)胞活性,降低骨質(zhì)疏松風(fēng)險(xiǎn),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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R580

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本文編號(hào)：2106408