CAJ | 학술논문

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0.8 mm/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（0.1 μg/μ1）重组质粒plRES-hVEGF165-hBMP2、pIRES-hBMP2、plRES-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Cyclins A、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 Cyclin A、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，14 d下降，28 d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14 d和28 d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。
목적 탐색전천공개도적기인치료대토하합골견인성골과정중세포주기조절단백표체적영향。방법 45지신서란대백토，쌍측하합골절골후3d개시이0.8 mm/d속도행하합골견인，련속견인7d후，수궤분위A、B、C、D、E5조，매조9지，분별재견인구주사2μg（0.1 μg/μ1）중조질립plRES-hVEGF165-hBMP2、pIRES-hBMP2、plRES-hVEGF165、공질립pIRES화상동제량적생리염수후，균시가전천공자격。각조분별우고정기제7、14、28천처사동물취재，행면역조직화학검사세포주기단백Cyclins A、D1、E적표체정황，병이용CMIAS-2001A병리도상분석계통분석，결과채용단인소방차분석화q검험。결과 Cyclin A、D1、E주요재육아조직중적염성세포여단핵세포、성섬유세포급소량연견장방향배렬적신생유치골소량표면적성골세포、골세포화골주위결체조직중표체；고정7d시표체최강렬，14 d하강，28 d시표체교약。도상분석결과현시，고정7d시C조양성표체단백적흡광도A치(0.59 +0.14)표체교강，여A(0.41±0.13)、B(0.38 +0.14)、D(0.34±0.12)、E(0.31 +0.10)조비교차이유통계학의의(P ＜0.05，P＜0.01)；A、B조간비교차이무통계학의의(P＞0.05)，단여D、E조비교차이유통계학의의(P＜0.05)；고정14 d화28 d시，A(0.39±0.11)、B(0.34±0.10)、C(0.33 +0.09)조간비교차이무통계학의의(P＞0.05)，단여D(0.19±0.12)、E(0.14 +0.04)조비교차이유통계학의의(P ＜0.05，P＜0.01)。각시상점기인치료조명현강우대조조。결론 전천공개도적기인치료능사세포주기단백CyclinsA、D1、E재견인구적표체증강、시한연장，가능촉진세포적분렬증식여분화，촉진견인구세포기질적형성화신골생성。
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. 1 μg/μl) of pIRES-hVEGF165-hBMP2, recombinant plasmid pIRES-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, electroporation 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 immunohistochemical 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 ceils,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 transfection can promote cyclins A, D1 ,E expression effectively, which may promote cell differentiation and proliferation, stimulate extracellular matrix synthesis and new bone formation in distraction gap.