电穿孔介导的基因治疗对兔下颌骨牵引过程中细胞周期蛋白表达的影响

Effect of electroporation-mediated gene transfect on the expression of cyclins during mandible distraction in rabbit

目的探索电穿孔介导的基因治疗对兔下颌骨牵引成骨过程中细胞周期调节蛋白表达的影响。方法45只新西兰大白兔，双侧下颌骨截骨后3d开始以0．8mm／d速度行下颌骨牵引，连续牵引7d后，随机分为A、B、c、D、E5组，每组9只，分别在牵引区注射2μg（0．1μg／μl）重组质粒pIRES-hVEGF165-hBMP2、pIRES—hBMP2、pIRES—hVEGF165、空质粒pIRES和相同剂量的生理盐水后，均施加电穿孔刺激。各组分别于固定期第7、14、28天处死动物取材，行免疫组织化学检查细胞周期蛋白CyclinsA、D1、E的表达情况，并利用CMIAS-2001A病理图像分析系统分析，结果采用单因素方差分析和q检验。结果CyclinA、D1、E主要在肉芽组织中的炎性细胞如单核细胞、成纤维细胞及少量沿牵张方向排列的新生幼稚骨小梁表面的成骨细胞、骨细胞和骨周围结缔组织中表达；固定7d时表达最强烈，14d下降，28d时表达较弱。图像分析结果显示，固定7d时C组阳性表达蛋白的吸光度A值（0．59±0．14）表达较强，与A（0．41±0．13）、B（0．38±0．14）、D（0．34±0．12）、E（0．31±0．10）组比较差异有统计学意义（P〈0．05，P〈0．01）；A、B组间比较差异无统计学意义（P〉0．05），但与D、E组比较差异有统计学意义（P〈0．05）；固定14d和28d时，A（0．39±0．11）、B（0．34±0．10）、c（0．33±0．09）组间比较差异无统计学意义（P〉0．05），但与D（0．19±0．12）、E（0．14±0．04）组比较差异有统计学意义（P〈0．05，P〈0．01）。各时相点基因治疗组明显强于对照组。结论电穿孔介导的基因治疗能使细胞周期蛋白CyclinsA、D1、E在牵引区的表达增强、时限延长，可能促进细胞的分裂增殖与分化，促进牵引区细胞基质的形成和新骨生成。

Objective To investigate the effect of electroporation-mediated gene transfect on the expression of cyclins during mandible distraction in rabbit. Methods Bilateral mandibular osteotomy was performed in 45 New-Zeland rabbits. After a latency of 3 days, the mandibles were elongated using distractors with a rate of 0. 8 mm/day for 7 days. After the completion of distraction, the rabbits were randomly divided into 5 groups. 2 μg（0.1μg/μl） of pIRES-hVEGF165-hBMP2, recombinant plasmid plRES-hBMP2, recombinant plasmid pIRES-hVEGF165, pIRES and the same volume of normal saline （NS） was injected into the distraction area in each group, respectively. After injection, eleetroporation was performed in every group. Three animals in each group were sacrificed at 7, 14, and 28 days after completion of distraction, respectively. The lengthened mandibles were harvested and processed for immunohistoehemical examinations. The expression of cyclins A, D1,E in positive cells were measured by CMIAS-2001A computerized image analyzer. The data were analyzed with the single factor analysis of variance and q test. Results Cyclins A, D1, E staining was mainly located in inflammatory cells, granulation tissue monocyte, fibroblast, osteoblasts, osteocyte and the connective tissues arrounding the new bone. The expression reached to the peak at 7th day of consolidation, and decreased at 14th day,and weak at 28th day. Image analysis results showed that, at 7th day, the expression abosorbance A in group C （0.59±0.14） was the strongest, compared to group A（0.41 ±0.13）, B（0.38 ±0.14）, D（0.34 ±0. 12） and E（0.31± 0. 10） , showing a significant difference （P 〈 0.05, P 〈 0. 01 ）. There was no significance difference between group A and B （P 〉 0.05 ） , but the difference between group A/B and group D/E （P 〈0.05）. At 14th and 28th day, there was no significant difference among group A（0.39 ± 0. 11）, B（0.34±0. 10） and C（0.33±0.09） （P〉0.05）, but there was significant difference between group A/B/C and group D （0. 19 ±0. 12） or E （0. 14±0.04） （P 〈0.05 or P 〈0.01）. Conclusions Electroporation-mediated gene transfeetion can promote cyclins A, D1 , E expression effectively, which may promote cell differentiation and proliferation, stimulate extraeellular matrix synthesis and new bone formation in distraction gap.