摘要
第一部分外源性Mfn2基因表达抑制平滑肌源性泡沫细胞形成
1.目的
研究平滑肌源性泡沫细胞内外源性Mfn2基因表达水平对于泡沫细胞形成的影响，对细胞内胆固醇流出的影响，研究其可能存在的分子机制。
2.方法
收集新鲜血清，采用密度梯度离心法提取LDL，铜离子氧化为ox-LDL后，与平滑肌细胞共培养24h或者48h，油红O染色观察泡沫细胞形成，体外构建平滑肌源性泡沫细胞模型。采用不同滴度水平的Adv-Mfn2（20pfu/cell、40pfu/cell和60pfu/cell）干预平滑肌细胞模型24h后，观察泡沫细胞形成的变化情况；酶化学法检测细胞内总胆固醇和游离胆固醇含量；采用WesternBlot方法检测胆固醇外转运蛋白ABCA1、ABCG1及核受体蛋白PPARγ蛋白磷酸化水平。
3.结果
重组腺病毒Adv-Mfn2（60pfu/cell）转染到平滑肌细胞内作用24小时后，平滑肌源性泡沫细胞脂滴明显减少，酶化学法检测证实细胞内总胆固醇含量下降，胆固醇酯/总胆固醇比例明显下降。WesternBlot证实重组腺病毒Adv-Mfn2干预组PPARγ蛋白磷酸化水平下降，胆固醇转运蛋白ABCA1、ABCG1表达水平上调。
4.结论
细胞内外源Mfn2基因在在适当水平的表达可以干预泡沫细胞内胆固醇的蓄积从而可以降低泡沫细胞形成，这一机制是通过调节PPARγ磷酸化活性及其下游的胆固醇外转运蛋白ABCA1和ABCG1的表达来实现。
第二部分外源性Mfn2基因对泡沫细胞形成丝裂原活化蛋白激酶信号通路的影响
1.目的
研究外源性Mfn2基因在平滑肌细胞内不同表达水平对于平滑肌源性泡沫细胞内与PPARγ活性相关的丝裂原活化蛋白激酶（MAPKs）信号通路的影响，研究外源性Mfn2基因抑制平滑肌源性泡沫细胞形成的分子信号通路。
2.方法
利用不同滴度水平重组腺病毒Adv-Mfn2（20pfu/cell、40pfu/cell和60pfu/cell）转染大鼠主动脉平滑肌细胞24h，然后加入ox-LDL与平滑肌细胞共培养48小时，采用WesternBlot检测ERK1/2、JNK、p38MAPKs信号通路蛋白磷酸化水平，统计学分析上述蛋白磷酸化水平的差异。3.结果
WesternBlot结果显示重组腺病毒Adv-mfn2在60pfu/cell水平转染大鼠平滑肌细胞条件下，ERK1/2、p38蛋白磷酸化水平明显下调，而与JNK磷酸化水平无关。
4.结论
外源性Mfn2基因在平滑肌细胞内适度表达水平能抑制泡沫细胞的形成，这一机制是通过抑制与PPARγ活性相关MAPK相关信号通路中的ERK1/2、p38信号通路蛋白磷酸化水平来实现，而与JNK信号通路无关。
PartⅠ The mechanism of Mfn2gene inhibits foam cell formation
1. Purpose
To explore the effect of expression of exogenous Mitofusin2(Mfn2) levels on rats vascluar smooth muscle-derived foam cells and the molecular mechanism of cellular cholesterol efflux that may exist.
2. Methods
The flesh serum of healthy individuals were collected and oxidated by copper ion after seperated LDL by density gradient centrifugation. Rats vascluar smooth muscle cells (rVSMCs) were co-cultured with oxidated LDL in vitro to induct the formation of foam cell, we make the intervention in different concentration of Adv-Mfn2(20pfu/cell,40pfu/cell, and60pfu/cell) in the induction of rVSMCs derived foam cells model. Foam cells were stained with oil red O and observed under a light microscope. The cholesterol level of foam cells were detected in the enzyme-chemical method and the ratio of total cholesterol to free cholesterol were calculated. The Cholesterol transporters adenosine trip hosphate-binding cassette subfamily A member1(ABCAl) and adenosine triphosphate-binding cassette subfamily G member1(ABCGl)and the phosphorylation of PPARy protein level were dectect by Western blot.
3. Results
While the rVSMCs were transfected with Adv-Mfn2(60pfu/cell) for24h with oxidated ldl(80ug/mL), the lipid droplets in foam cells were reduced significantly. The ratio of intracellular free cholesterol ester compared to total cholesterol levels dropped significantly detected by chemical-enzyme assay. The phosphorylation level of PPARy protein decreased and the expression level of ABCA1and ABCG1increased detected by Western blot.
4. Conclusions
Adv-Mfn2could significantly reduce the formation of foam cells after transfected at the appropriate level, and furthermore it could decreases the intracellular cholesterol levels to prompt the expression of cholesterol transport protein ABCA1and ABCG1.
PartⅡ The effect of exogenous Mfn2gene on mitogen-activated protein kinase signaling pathway in foam cell formation
1. Purpose
To investigate the the mechanism of different expression concentration of mitofusin2gene on the phosphorylation level of MAPKs signaling pathway related to the formation of smooth muscle-derived foam cells.
2. Methods
rVSMCs were transfected different titers of adv-Mfn2(20,40and60pfu/cell) for24h, and then it were co-cultured with the ox-LDL and for24hours. The phosphorylation level of ERK1/2, JNK and p38signaling pathway proteins were detected by Western blot. Statistical significances were compared using Tukey-Kramer post hoc test and student t-test by SPSS12.0. Differences were considered significant when P<0.05.
3. Results
The Western Blot results showed that when foam cells were transfected with60pfu/cell, the phosphorylation levels of ERK1/2of p38proteins of the rVSMC derived foam cells were significantly reduced, regardless of the phosphorylation level of JNK.
4. Conclusions
The moderate level of exogenous Mfn2expression in rVSMC derived foam cells could inhibit the induction of foam cell formation with oxidiated ldl in vitro, which were achieved by reducing the the phosphorylation level of ERK1/2and p38signaling pathway, and independent of the phosphorylation level of JNK signaling pathway.
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