双基因重组载体预防兔乙醇性股骨头坏死的组织病理学观察

The histopathological observation to prevent ethanol-induced osteonecrosis of the femoral head of rabbits by double gene recombinant vector in vivo

目的观察双基因重组载体预防兔乙醇性股骨头坏死(ONFH)的组织病理学变化。方法自2018年1月至2019年6月将购自河南省实验动物中心的80只兔采用完全随机化原则分为5组:正常组(N,正常动物,空白对照)、致敏组(H,仅马血清致敏,不给予白酒)、模型组[M,马血清致敏,烈性白酒10 ml/(kg·d)灌胃]、无关序列组[Con,马血清致敏,白酒灌胃,一侧股骨头注入无关序列pGFP-V-RS转染的骨髓间充质干细胞(BMSCs)]、双基因组(S,马血清致敏,白酒灌胃,一侧股骨头内注入双基因重组载体pGFP-V-RS-siPPARγ-exCGRP转染的BMSCs)。实验第4、8周末分批处死动物,观察兔股骨头病理学改变。两样本两组间比较采用t检验;分类变量数据资料分析采用χ2检验;数值变量资料统计采用方差分析、组间比较采用最小显著性差异法(LSD)。结果实验第8周,S、H组中骨小梁面积分数分别为(40.80±3.16)、(41.20±3.65)%,与正常组[(41.80±3.63)%]比较差异无统计学意义(t=0.588、0.661,P>0.05),明显高于M组[(28.10±2.55)%]、Con组[(28.30±2.65)%],差异均有统计学意义(t=8.846、8.496、8.322、8.089,P<0.01)。S、H、N组中空骨陷窝计数分别为(13.30±1.66)、(13.10±1.63)、(12.30±1.46)%,组间比较差异均无统计学意义(t=0.243、1.279、1.273,P>0.05),明显低于M组[(39.30±3.86)%]、Con组[(39.10±3.65)%],差异均有统计学意义(t=17.502、18.397、17.686、18.397、18.505、19.282,P<0.01)。S、H组中最大脂肪细胞平均直径分别为(42.55±4.16)、(42.53±4.15)μm,与正常组[(41.36±3.85)μm]比较,差异均无统计学意义(t=0.594、0.577,P>0.05),明显低于M组[(52.31±5.26)μm]、Con组[(52.19±5.13)μm],差异均有统计学意义(t=4.116、4.128、4.129、4.141,P<0.01)。M、Con组中出现明显的早期ONFH病理学变化,股骨头软骨下骨区的造血组织显著减少,骨小梁变细稀疏、中断、结构紊乱,骨髓坏死,骨小梁面积分数降低、空骨陷窝显著增多,脂肪细胞增殖肥大,脂肪组织堆积。N组中无这些ONFH病理学改变。S、H组中病理学改变很少,骨组织结构接近于N组。8周时,M、Con组中病理学变化比4周时加重,ONFH发生率为87.5%、87.5%,而S、H、N组中为12.5%、12.5%、0%(χ2=9.833、9.833、12.444、9.833、9.833、12.444,P<0.01),差异有统计学意义。结论双基因重组载体能够高效阻止乙醇导致兔ONFH的组织病理学改变,预防乙醇性ONFH的发生,显著优于单一基因的作用。

Objective To observe the histopathological changes of ethanol-induced osteonecrosis of the femoral head(ONFH)in rabbits treated by double gene recombinant vector in vivo.Methods From January 2018 to June 2019,80 healthy rabbits purchased from the Experimental Animal Center of Henan Province were randomly divided into 5 groups:(1)normal group receiving no treatment;(2)sensitization group treated with horse serum sensitization only;(3)model group treated with 10 ml/(kg·d)of spirits containing 46%ethanol gavage after horse serum sensitization;(4)unrelated sequence group treated with unrelated sequence-transfected bone marrow mesenchymal stem cells(BMSCs)and 10 ml/(kg·d)of spirits containing 46%ethanol gavage after horse serum sensitization;(5)double gene group given double gene recombinant vector-transfected BMSCs injected into one-side femoral head of the rabbits and 10 ml/(kg·d)of spirits containing 46%ethanol gavage after horse serum sensitization.At the end of the 4th and 8th week,the animals were sacrificed in batches,and the pathological changes of femoral head were observed.T-test was used for comparison between the two groups.Data of categorical variables were analyzed by chi-square test;Analysis of variance was used for data statistics of numerical variables,and LSD method was used for comparison between groups.Results At the 8th week of the experiment,the bone trabecular area fractions in sensitization group and double gene group were(40.80±3.16)%and(41.20±3.65)%respectively,which were not significantly different from those in normal group[(41.80±3.63)%,t=0.588,0.661,P>0.05],but significantly higher than those in the model group[(28.10±2.55)%]and unrelated sequence group[(28.30±2.65)%,t=8.846,8.496,8.322,8.089,P<0.01].The percentages of empty osteocyte lacunae in sensitization group,double gene group and normal group were(13.30±1.66)%,(13.10±1.63)%and(12.30±1.46)%respectively,with the differences being not statistically significant among them(t=0.243,1.279,1.273,P>0.05),but significantly lower than those in model group[(39.30±3.86)%]and unrelated sequence group[(39.10±3.65)%],with the difference being statistically significant(t=17.502,18.397,17.686,18.397,18.505,19.282,P<0.01).The biggest adipocytes diameter in double gene group and sensitization group was(42.55±4.16)and(42.53±4.15)μm respectively,which was not significantly different from that in normal group[(41.36±3.85)μm,t=0.594,0.577,P>0.05],but significantly lower than that in model group[(52.31±5.26)μm]and unrelated sequence group[(52.19±5.13)μm,t=4.116,4.128,4.129,4.141,P<0.01].There were obviously early pathological changes of ONFH in model group and control group at bone area under cartilage,such as the hematopoiesis tissue in the subchondral bone area of the femoral head was significantly reduced,the bone trabecula became thin and sparse,interrupted,the structure was disordered,and there was necrosis of bone marrow in some regions.The bone trabecular area fractions were decreased,the empty bone lacuna was significantly increased,adipocyte proliferation was hypertrophy,and the fat tissue was accumulated.In normal group,above ONFH pathological changes were not seen.There were few pathological changes in sensitization group and double gene group,and the bone tissue structure was similar with that in normal group.At 8th week,the pathological changes in model and unrelated sequence groups were worse than those at 4th week,and the incidence rate of ONFH was 87.5%and 87.5%in model group and unrelated sequence group,and that was 12.5%,12.5%and 0%in double group,sensitization group and normal group,respectively(χ2=9.833,9.833,12.444,9.833,9.833,12.444,P<0.01).Conclusion The double gene recombinant vector can especially and effectively suppress the ethanol-induced pathological changes of ONFH in rabbits and prevent the occurrence of ethanol-induced ONFH,which is significantly better than that of a single gene.