目的 通过检测不同诱发途径建立的子痫前期模型孕鼠胎盘组织中长链脂肪酸β氧化关键酶长链-3-羟酰基辅酶A脱氢酶(LCHAD)基因位点甲基化程度的差异,探讨多因素和多通路在子痫前期病理机制中的分子实验基础.方法 按不同途径建立子痫前期孕鼠模型并分组如下:(1)左硝基精氨酸甲酯(L-NAME)组:给予孕鼠皮下注射L-NAME的方法建模;(2)脂多糖(LPS)组:给予孕鼠腹腔注射LPS的方法建模;(3)载脂蛋白C3(ApoC3)组:给予存在脂代谢障碍的孕鼠皮下注射L-NAME的方法建模型;(4)β2糖蛋白Ⅰ(β-2GPI)组:给予孕鼠皮下注射β-2GPI不完全弗氏佐剂的方法建模.根据建模时间对L-NAME组、LPS组及ApoC3组孕鼠于妊娠第3、11、16天分别定为胚胎植入前期、子痫前期早发期和晚发期3个不同实验阶段,β-2GPI组孕鼠仅有植入前期,对不同实验阶段的孕鼠胎盘LCHAD基因位点的甲基化水平进行检测.同时设生理盐水对照组,以及应用ApoC3高表达转基因孕鼠作为对照的转基因对照组.结果 (1)各组孕鼠LCHAD基因发生甲基化的位点:在LCHAD基因转录起始位点上游728 bp的范围内,共发现存在45个CG位点,其中LCHAD基因CG-5、12、13、14、15、16、19、24、25、27、28、29、30、31、32、34、35、43为含有2个及以上CG序列的复合位点,其他位点为仅含有1个CG序列的单一位点.L-NAME组、LPS组、ApoC3组和β-2GPI组孕鼠均有13个LCHAD基因位点发生甲基化改变,其中,LCHAD基因CG-3、5、6、11、13、14、18、28位点呈不同程度的高甲基化状态;LCHAD基因CG-16、25、31、42、44位点呈不同程度的低甲基化状态;其余32个位点无甲基化改变.(2)各组孕鼠胎盘LCHAD基因位点甲基化水平比较:L-NAME组、LPS组、ApoC3组、β-2GPI组和转基因对照组孕鼠胎盘LCHAD基因CG-3、11、13、14、18位点的甲基化水平显著高于生理盐水对照组,分别比较,差异均有统计学意义(P<0.05);而LCHAD基因CG-42、44位点的甲基化水平显著低于生理盐水对照组,分别比较,差异有统计学意义(P<0.05).(3)各组孕鼠LCHAD基因相同位点的甲基化水平比较:①L-NAME组孕鼠早发期LCHAD基因CG-3、11、18位点的甲基化水平显著低于植入前期及晚发期,LPS组孕鼠植入前期显著低于早发期及晚发期;而ApoC3组孕鼠植入前显著高于早发期及晚发期,分别比较,差异均有统计学意义(P<0.05).②L-NAME组、LPS组不同实验阶段孕鼠LCHAD基因CG-5位点甲基化水平均显著高于生理盐水对照组,而ApoC3组仅植入前及早发期高于生理盐水对照组,分别比较,差异均有统计学意义(P<0.05).③L-NAME组孕鼠不同实验阶段LCHAD基因CG-6位点甲基化水平显著高于其他各组,分别比较,差异均有统计学意义(P<0.05).④L-NAME组孕鼠LCHAD基因CG-13、14位点甲基化水平在植入前、早发期、晚发期呈逐渐升高趋势,LPS组孕鼠在植入前显著高于早发期、晚发期,而ApoC3组孕鼠早发期的甲基化水平最高,分别比较,差异均有统计学意义(P<0.05).⑤L-NAME组孕鼠LCHAD基因CG-28位点甲基化水平在植入前、早发期、晚发期呈逐渐降低趋势,LPS组孕鼠早发期、晚发期甲基化水平高于植入前期,ApoC3组孕鼠早发期甲基化水平最高,分别比较,差异均有统计学意义(P<0.05).⑥ApoC3组孕鼠LCHAD基因CG-16、25、31位点甲基化水平显著高于其他各组,分别比较,差异均有统计学意义(P<0.05).⑦β-2GPI组孕鼠LCHAD基因CG-42位点未检测到甲基化,而其他各组CG-42位点呈低甲基化状态,β-2GPI组与其他各组的CG-42位点甲基化水平比较,差异均有统计学意义(P<0.05).结论 LCHAD基因CG-6、42位点甲基化可能分别与L-NAME、β-2GPI诱导的子痫前期孕鼠胎盘中LCHAD基因表达变化相关;LCHAD基因表达和甲基化在LPS和ApoC3转基因孕鼠中未见明显联系.不同诱发途径的子痫前期孕鼠LCHAD甲基化水平和表达存在的差异,进一步揭示了多因素和多通路在子痫前期发病机制中的分子实验基础.
目的 通過檢測不同誘髮途徑建立的子癇前期模型孕鼠胎盤組織中長鏈脂肪痠β氧化關鍵酶長鏈-3-羥酰基輔酶A脫氫酶(LCHAD)基因位點甲基化程度的差異,探討多因素和多通路在子癇前期病理機製中的分子實驗基礎.方法 按不同途徑建立子癇前期孕鼠模型併分組如下:(1)左硝基精氨痠甲酯(L-NAME)組:給予孕鼠皮下註射L-NAME的方法建模;(2)脂多糖(LPS)組:給予孕鼠腹腔註射LPS的方法建模;(3)載脂蛋白C3(ApoC3)組:給予存在脂代謝障礙的孕鼠皮下註射L-NAME的方法建模型;(4)β2糖蛋白Ⅰ(β-2GPI)組:給予孕鼠皮下註射β-2GPI不完全弗氏佐劑的方法建模.根據建模時間對L-NAME組、LPS組及ApoC3組孕鼠于妊娠第3、11、16天分彆定為胚胎植入前期、子癇前期早髮期和晚髮期3箇不同實驗階段,β-2GPI組孕鼠僅有植入前期,對不同實驗階段的孕鼠胎盤LCHAD基因位點的甲基化水平進行檢測.同時設生理鹽水對照組,以及應用ApoC3高錶達轉基因孕鼠作為對照的轉基因對照組.結果 (1)各組孕鼠LCHAD基因髮生甲基化的位點:在LCHAD基因轉錄起始位點上遊728 bp的範圍內,共髮現存在45箇CG位點,其中LCHAD基因CG-5、12、13、14、15、16、19、24、25、27、28、29、30、31、32、34、35、43為含有2箇及以上CG序列的複閤位點,其他位點為僅含有1箇CG序列的單一位點.L-NAME組、LPS組、ApoC3組和β-2GPI組孕鼠均有13箇LCHAD基因位點髮生甲基化改變,其中,LCHAD基因CG-3、5、6、11、13、14、18、28位點呈不同程度的高甲基化狀態;LCHAD基因CG-16、25、31、42、44位點呈不同程度的低甲基化狀態;其餘32箇位點無甲基化改變.(2)各組孕鼠胎盤LCHAD基因位點甲基化水平比較:L-NAME組、LPS組、ApoC3組、β-2GPI組和轉基因對照組孕鼠胎盤LCHAD基因CG-3、11、13、14、18位點的甲基化水平顯著高于生理鹽水對照組,分彆比較,差異均有統計學意義(P<0.05);而LCHAD基因CG-42、44位點的甲基化水平顯著低于生理鹽水對照組,分彆比較,差異有統計學意義(P<0.05).(3)各組孕鼠LCHAD基因相同位點的甲基化水平比較:①L-NAME組孕鼠早髮期LCHAD基因CG-3、11、18位點的甲基化水平顯著低于植入前期及晚髮期,LPS組孕鼠植入前期顯著低于早髮期及晚髮期;而ApoC3組孕鼠植入前顯著高于早髮期及晚髮期,分彆比較,差異均有統計學意義(P<0.05).②L-NAME組、LPS組不同實驗階段孕鼠LCHAD基因CG-5位點甲基化水平均顯著高于生理鹽水對照組,而ApoC3組僅植入前及早髮期高于生理鹽水對照組,分彆比較,差異均有統計學意義(P<0.05).③L-NAME組孕鼠不同實驗階段LCHAD基因CG-6位點甲基化水平顯著高于其他各組,分彆比較,差異均有統計學意義(P<0.05).④L-NAME組孕鼠LCHAD基因CG-13、14位點甲基化水平在植入前、早髮期、晚髮期呈逐漸升高趨勢,LPS組孕鼠在植入前顯著高于早髮期、晚髮期,而ApoC3組孕鼠早髮期的甲基化水平最高,分彆比較,差異均有統計學意義(P<0.05).⑤L-NAME組孕鼠LCHAD基因CG-28位點甲基化水平在植入前、早髮期、晚髮期呈逐漸降低趨勢,LPS組孕鼠早髮期、晚髮期甲基化水平高于植入前期,ApoC3組孕鼠早髮期甲基化水平最高,分彆比較,差異均有統計學意義(P<0.05).⑥ApoC3組孕鼠LCHAD基因CG-16、25、31位點甲基化水平顯著高于其他各組,分彆比較,差異均有統計學意義(P<0.05).⑦β-2GPI組孕鼠LCHAD基因CG-42位點未檢測到甲基化,而其他各組CG-42位點呈低甲基化狀態,β-2GPI組與其他各組的CG-42位點甲基化水平比較,差異均有統計學意義(P<0.05).結論 LCHAD基因CG-6、42位點甲基化可能分彆與L-NAME、β-2GPI誘導的子癇前期孕鼠胎盤中LCHAD基因錶達變化相關;LCHAD基因錶達和甲基化在LPS和ApoC3轉基因孕鼠中未見明顯聯繫.不同誘髮途徑的子癇前期孕鼠LCHAD甲基化水平和錶達存在的差異,進一步揭示瞭多因素和多通路在子癇前期髮病機製中的分子實驗基礎.
목적 통과검측불동유발도경건립적자간전기모형잉서태반조직중장련지방산β양화관건매장련-3-간선기보매A탈경매(LCHAD)기인위점갑기화정도적차이,탐토다인소화다통로재자간전기병리궤제중적분자실험기출.방법 안불동도경건립자간전기잉서모형병분조여하:(1)좌초기정안산갑지(L-NAME)조:급여잉서피하주사L-NAME적방법건모;(2)지다당(LPS)조:급여잉서복강주사LPS적방법건모;(3)재지단백C3(ApoC3)조:급여존재지대사장애적잉서피하주사L-NAME적방법건모형;(4)β2당단백Ⅰ(β-2GPI)조:급여잉서피하주사β-2GPI불완전불씨좌제적방법건모.근거건모시간대L-NAME조、LPS조급ApoC3조잉서우임신제3、11、16천분별정위배태식입전기、자간전기조발기화만발기3개불동실험계단,β-2GPI조잉서부유식입전기,대불동실험계단적잉서태반LCHAD기인위점적갑기화수평진행검측.동시설생리염수대조조,이급응용ApoC3고표체전기인잉서작위대조적전기인대조조.결과 (1)각조잉서LCHAD기인발생갑기화적위점:재LCHAD기인전록기시위점상유728 bp적범위내,공발현존재45개CG위점,기중LCHAD기인CG-5、12、13、14、15、16、19、24、25、27、28、29、30、31、32、34、35、43위함유2개급이상CG서렬적복합위점,기타위점위부함유1개CG서렬적단일위점.L-NAME조、LPS조、ApoC3조화β-2GPI조잉서균유13개LCHAD기인위점발생갑기화개변,기중,LCHAD기인CG-3、5、6、11、13、14、18、28위점정불동정도적고갑기화상태;LCHAD기인CG-16、25、31、42、44위점정불동정도적저갑기화상태;기여32개위점무갑기화개변.(2)각조잉서태반LCHAD기인위점갑기화수평비교:L-NAME조、LPS조、ApoC3조、β-2GPI조화전기인대조조잉서태반LCHAD기인CG-3、11、13、14、18위점적갑기화수평현저고우생리염수대조조,분별비교,차이균유통계학의의(P<0.05);이LCHAD기인CG-42、44위점적갑기화수평현저저우생리염수대조조,분별비교,차이유통계학의의(P<0.05).(3)각조잉서LCHAD기인상동위점적갑기화수평비교:①L-NAME조잉서조발기LCHAD기인CG-3、11、18위점적갑기화수평현저저우식입전기급만발기,LPS조잉서식입전기현저저우조발기급만발기;이ApoC3조잉서식입전현저고우조발기급만발기,분별비교,차이균유통계학의의(P<0.05).②L-NAME조、LPS조불동실험계단잉서LCHAD기인CG-5위점갑기화수평균현저고우생리염수대조조,이ApoC3조부식입전급조발기고우생리염수대조조,분별비교,차이균유통계학의의(P<0.05).③L-NAME조잉서불동실험계단LCHAD기인CG-6위점갑기화수평현저고우기타각조,분별비교,차이균유통계학의의(P<0.05).④L-NAME조잉서LCHAD기인CG-13、14위점갑기화수평재식입전、조발기、만발기정축점승고추세,LPS조잉서재식입전현저고우조발기、만발기,이ApoC3조잉서조발기적갑기화수평최고,분별비교,차이균유통계학의의(P<0.05).⑤L-NAME조잉서LCHAD기인CG-28위점갑기화수평재식입전、조발기、만발기정축점강저추세,LPS조잉서조발기、만발기갑기화수평고우식입전기,ApoC3조잉서조발기갑기화수평최고,분별비교,차이균유통계학의의(P<0.05).⑥ApoC3조잉서LCHAD기인CG-16、25、31위점갑기화수평현저고우기타각조,분별비교,차이균유통계학의의(P<0.05).⑦β-2GPI조잉서LCHAD기인CG-42위점미검측도갑기화,이기타각조CG-42위점정저갑기화상태,β-2GPI조여기타각조적CG-42위점갑기화수평비교,차이균유통계학의의(P<0.05).결론 LCHAD기인CG-6、42위점갑기화가능분별여L-NAME、β-2GPI유도적자간전기잉서태반중LCHAD기인표체변화상관;LCHAD기인표체화갑기화재LPS화ApoC3전기인잉서중미견명현련계.불동유발도경적자간전기잉서LCHAD갑기화수평화표체존재적차이,진일보게시료다인소화다통로재자간전기발병궤제중적분자실험기출.
Objective By detecting the variation of long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) DNA methylation in preeclampsia-like mouse models generated by different ways, to explore the roles of multifactor and multiple pathways in preeclampsia pathogenesis on molecular basis. Methods Established preeclampsia-like mouse models in different ways and divided into groups as follows: (1) Nw-nitro-L-arginine-methyl ester (L-NAME) group: wild-type pregnant mouse received subcutaneous injection of L-NAME;(2) lipopolysaccharide (LPS) group:wild-type pregnant mouse received intraperitoneal injection of LPS; (3) apolipoprotein C-Ⅲ (ApoC3) group: ApoC3 transgenic pregnant mouse with dysregulated lipid metabolism received subcutaneous injection of L-NAME;(4)β2 glycoprotein I (β-2GPI) group:wild-type pregnant mouse received subcutaneous injection ofβ-2GPI. According to the first injection time (on day 3, 11, 16 respectively), the L-NAME, LPS and ApoC3 groups were further subdivided into:pre-implantation (PI) experimental stage, early gestation (EG) experimental stage, and late gestation (LG) experimental stage.β-2GPI group was only injected before implantation. LCHAD gene methylation levels in placental were detected in different experimental stage. Normal saline control groups were set within wild-type and ApoC3 transgenic pregnant mice simultaneously. Results (1) CG sites in LCHAD DNA:45 CG sites were detected in the range of 728 bp before LCHAD gene transcription start site, the 5, 12, 13, 14, 15, 16, 19, 24, 25, 27, 28, 29, 30, 31, 32, 34, 35, 43 CG sites were complex sites which contained two or more CG sequences, others were single site which contained one CG sequence. The 3, 5, 6, 11, 13, 14, 18, 28 sites in L-NAME, LPS, ApoC3 and β-2GPI groups showed different high levels of methylation; the 16, 25, 31, 42, 44 sites showed different low levels of methylation; other 32 sites were unmethylated. (2) Comparison of LCHAD gene methylation between different groups:the methylation levels of LCAHD gene at 3, 11, 13, 14, 18 sites in L-NAME, LPS, ApoC3 andβ-2GPI groups were significantly higher than those in the normal saline control group (P<0.05); and the methylation levels of 42, 44 sites in these groups were significantly lower than those in the normal saline control group (P<0.05). (3) Methylation of LCHAD gene at the same site between different experimental stages: ① The 3, 11, 18 sites of EG experimental stage was significantly lower than PI and LG experimental stage in L-NAME group (P<0.05);the 3, 11, 18 sites of PI experimental stage was significantly lower than EG and LG experimental stage in LPS group (P<0.05);these sites of PI experimental stage was significantly higher than EG and LG experimental stages in ApoC3 group (P<0.05).②The methylation of site 5 in L-NAME and LPS groups were significantly higher than that of the normal saline control group (P<0.05), and the LG experimental stages were significantly higher than other stages, but in ApoC3 group , only PI and EG stages were significantly higher than the normal saline control group (P<0.05).③At site 6 in L-NAME group which showed high methylation level was significantly higher than the same site in other groups which showed low methylation level (P<0.05).④At 13, 14 sites, earlier preeclampsia onset caused a lower methylation level in L-NAME group, but PI experimental stage was significantly higher than EG and LG experimental stages in LPS group (P<0.05), EG experimental stage was significantly higher than PI and LG experimental stages in ApoC3 group (P<0.05). ⑤ At site 28, earlier preeclampsia onset caused a higher methylation level in L-NAME group, but PI experimental stage was significantly lower than EG and LG experimental stages in LPS group (P<0.05), EG experimental stage was significantly higher than PI and LG experimental stages in ApoC3 group (P<0.05).⑥The 16, 25, 31 sites in ApoC3 group were significantly higher than other groups (P<0.05). ⑦ At site 42 in β-2GPI group was unmethylated, but it in other groups showed low methylation level, the methylation level of site 42 inβ-2GPI group was significantly lower than that in other groups (P<0.05). Conclusions The methylation of 6 and 42 CG sites may be related to LCHAD gene expression in placenta of L-NAME and β-2GPI induced preeclampsia-like models respectively;LCHAD gene expression and DNA methylation may not have obviouscorrelation in LPS and ApoC3 induced preeclampsia-like models. Differences exist in LCHAD DNA methylation in preeclampsia-like models generated by different ways, revealed a molecular basis to expand our understanding of the multi-factorial pathogenesis of preeclampsia.
