RNAi技术抑制同源异型盒基因HLX1表达对滋养细胞增殖和侵袭的影响
发布时间：2018-08-16 09:04
【摘要】： 【研究背景及目的】 同源异型盒基因是一类重要的调控细胞分化和形态发育的转录因子基因家族,其中HLX1基因与胚胎来源细胞和造血系细胞的增殖、分化行为密切相关。近年来研究陆续表明,HLX1基因对于正常胎盘形成起重要作用;HLX1蛋白在细胞滋养细胞来源的细胞系HTR-8/SVneo、SGHPL-4、JEG-3、JAR和BeWo的细胞中均有表达;病理妊娠如特发性胎儿宫内生长受限(FGR)的胎盘中HLX1基因表达水平明显降低。本研究旨在运用RNAi技术抑制滋养细胞株HTR-8/SVneo中HLX1基因的表达,探讨其对体外滋养细胞增殖和侵袭行为的影响,为HLX1基因异常表达可能参与FGR等病理妊娠的发病提供基础理论支持。 【方法】 1.复苏、培养人早孕期胎盘绒毛膜外滋养细胞株HTR-8/SVneo至对数生长期备用。 2.应用反转录-聚合酶链反应(RT-PCR)、Western Blot方法检测HTR-8/SVneo细胞中HLX1 mRNA和蛋白质的表达。 3.依据GeneBank中HLX1 cDNA序列号NM_021958,设计并化学合成siRNA序列,分别为:4条特异性HLX1 siRNA的预混试剂;1条非特异性阴性对照siRNA试剂,DY-547荧光标记。 4.HTR-8/SVneo细胞常规体外培养,设为三组:实验组(转染HLX1 siRNAs)、阴性对照组(转染阴性对照siRNA)以及空白对照组(除不含siRNA,余试剂与另两组均相同),分别进行瞬时转染。 5.将DY-547标记的阴性对照siRNA转染入HTR-8/SVneo细胞,应用流式细胞术(FCM)测定转染效率,筛选出转染效率最佳优化条件进行后续实验。 6.应用实时荧光定量PCR(qRT-PCR)和Western Blot方法分别检测三组细胞转染后HLX1基因mRNA和蛋白的表达,评价干扰效果。 7.细胞增殖实验:将三组HTR-8/SVneo细胞以相同数量接种,过夜培养后进行瞬时转染,应用四甲基偶氮唑盐(MTT)比色法每隔24测定吸光度值后绘制生长曲线。 8.细胞侵袭实验:应用Transwell小室检测转染后各组HTR-8/SVneo细胞穿透人工重组基底膜的能力。三组HTR-8/SVneo细胞以相同数量接种,过夜培养后进行瞬时转染,24小时后,用无血清培养液重悬细胞并以相同数量接种于包被有Matrigel基质胶的小室上室,下室加入20%FCS的培养液。继续培养24h,40倍镜下随机取5个视野计数穿膜细胞数。 9.统计学方法:应用SPSS 11.0软件包进行统计分析,以均数±标准差((?)±s)表示定量资料数据,组间比较采用单因素方差分析(ANOVA),认为P＜0.05有统计学意义。 【结果】 1.HLX1 mRNA和蛋白在HTR-8/SVneo细胞中均呈阳性表达。 2.siRNA转染效率优化后可达(86.3±2.6)%,以相同条件进行后续实验。 3.与空白对照组及阴性对照组相比,实验组转染HLX1 siRNAs后特异且有效地抑制了HTR-8/SVneo细胞中HLX1基因的表达,转染48h后HLX1 mRNA的表达降低(77.0±1.2)%(P＜0.01),转染72h后HLX1蛋白的抑制率为(82.6±1.2)%(P＜0.01)。 4.与空白对照组及阴性对照组相比,实验组转染HLX1 siRNAs后HTR-8/SVneo细胞的增殖活性下降,转染72h后增殖抑制效应最为显著,抑制率为(58.1±4.4)%(P＜0.01)。 5.与空白对照组及阴性对照组相比,实验组转染HLX1 siRNAs后HTR-8/SVneo细胞穿过Matrigel基质胶的侵袭能力受到显著抑制,差异有统计学意义(P＜0.01)。 【结论】 HLX1 siRNAs可有效抑制人早孕期胎盘绒毛膜外滋养细胞HTR-8/SVneo中HLX1基因的表达,HLX1基因表达下降可以显著抑制滋养细胞的增殖活性和侵袭能力,它的表达异常可能通过影响滋养细胞增殖、侵袭等生物学性能而在FGR等病理妊娠的发病中起重要作用。
[Abstract]:[background and purpose]
HLX1 gene is closely related to the proliferation and differentiation of embryonic and hematopoietic cells. Recent studies have shown that HLX1 gene plays an important role in normal placenta formation; HLX1 protein plays an important role in cytotrophoblast formation. The expression of HLX1 gene in the placenta of pathological pregnancies such as idiopathic fetal growth restriction (FGR) was significantly decreased. The aim of this study was to investigate the effect of RNAi on the expression of HLX1 gene in the trophoblastic cell line HTR-8/SVneo, and to investigate its effect on trophoblast cells in vitro. The effects of reproductive and invasive behaviors provide basic theoretical support for the abnormal expression of HLX1 gene in FGR and other pathological pregnancies.
[method]
1. resuscitation, culture human placental chorionic villus trophoblastic strain HTR-8/SVneo from early pregnancy to logarithmic growth phase.
2. Reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the expression of HLX1 mRNA and protein in HTR-8/SVneo cells.
3. According to the sequence number NM_021958 of HLX1 cDNA in GeneBank, the siRNA sequences were designed and synthesized. They were: 4 pre-mixing reagents for specific HLX1 siRNA, 1 non-specific negative control reagent, DY-547 fluorescent labeling.
4. HTR-8/SVneo cells were routinely cultured in vitro and divided into three groups: experimental group (transfected with HLX1 siRNAs), negative control group (transfected with negative control siRNA) and blank control group (except without siRNA, other reagents were the same as the other two groups), and transfected instantaneously.
5. DY-547-labeled negative control siRNA was transfected into HTR-8/SVneo cells. The transfection efficiency was determined by flow cytometry (FCM), and the optimum conditions for transfection efficiency were screened out.
6. Real-time fluorescence quantitative PCR (qRT-PCR) and Western Blot were used to detect the expression of HLX1 gene mRNA and protein in three groups of cells, respectively, to evaluate the interference effect.
7. Cell proliferation experiment: Three groups of HTR-8/SVneo cells were inoculated in the same amount and transfected instantaneously after overnight culture. The growth curve was drawn by MTT colorimetry every 24 hours.
8. Cell invasion assay: The ability of HTR-8/SVneo cells to penetrate the artificially reconstituted basement membrane was detected by Transwell chamber. Three groups of HTR-8/SVneo cells were inoculated in the same amount, and were transfected instantly after overnight culture. After 24 hours, the cells were suspended in serum-free medium and inoculated with Matrigel matrix gum in the same amount. In the upper compartment, 20% FCS medium was added to the lower compartment. After 24 hours of culture, the number of penetrating cells was counted by 5 visual fields randomly under 40-fold microscope.
9. Statistical methods: SPSS 11.0 software package was used for statistical analysis, and the mean (?) + standard deviation (?) + s) was used for quantitative data. One-way ANOVA was used for inter-group comparison, and P < 0.05 was statistically significant.
[results]
1.HLX1 mRNA and protein were positive in HTR-8/SVneo cells.
The transfection efficiency of 2.siRNA reached (86.3 + 2.6)%, followed by the same conditions.
3. Compared with the blank control group and the negative control group, the experimental group specifically and effectively inhibited the expression of HLX1 gene in HTR-8/SVneo cells after transfection of HLX1 siRNAs. After 48 hours of transfection, the expression of HLX1 mRNA was decreased (77.0 (+ 1.2)) (P < 0.01), and the inhibition rate of HLX1 protein was (82.6 (+ 1.2)) (P < 0.01).
4. Compared with the blank control group and the negative control group, the proliferation activity of HTR-8/SVneo cells in the experimental group decreased after transfection of HLX1 siRNAs, and the proliferation inhibition effect was the most significant 72 hours after transfection, the inhibition rate was (58.1+4.4)% (P < 0.01).
5. Compared with the blank control group and the negative control group, the invasive ability of HTR-8/SVneo cells transfected with HLX1 siRNAs was significantly inhibited (P<0.01).
[Conclusion]
HLX1 siRNAs can effectively inhibit the expression of HLX1 gene in human placental extrachorionic trophoblast HTR-8/SVneo during early pregnancy. The decrease of HLX1 gene expression can significantly inhibit the proliferation and invasiveness of human placental extrachorionic trophoblasts. The abnormal expression of HLX1 may affect the proliferation and invasiveness of trophoblasts in FGR and other pathological pregnancy. Plays an important role.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346;R714.2
[Abstract]:[background and purpose]
HLX1 gene is closely related to the proliferation and differentiation of embryonic and hematopoietic cells. Recent studies have shown that HLX1 gene plays an important role in normal placenta formation; HLX1 protein plays an important role in cytotrophoblast formation. The expression of HLX1 gene in the placenta of pathological pregnancies such as idiopathic fetal growth restriction (FGR) was significantly decreased. The aim of this study was to investigate the effect of RNAi on the expression of HLX1 gene in the trophoblastic cell line HTR-8/SVneo, and to investigate its effect on trophoblast cells in vitro. The effects of reproductive and invasive behaviors provide basic theoretical support for the abnormal expression of HLX1 gene in FGR and other pathological pregnancies.
[method]
1. resuscitation, culture human placental chorionic villus trophoblastic strain HTR-8/SVneo from early pregnancy to logarithmic growth phase.
2. Reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the expression of HLX1 mRNA and protein in HTR-8/SVneo cells.
3. According to the sequence number NM_021958 of HLX1 cDNA in GeneBank, the siRNA sequences were designed and synthesized. They were: 4 pre-mixing reagents for specific HLX1 siRNA, 1 non-specific negative control reagent, DY-547 fluorescent labeling.
4. HTR-8/SVneo cells were routinely cultured in vitro and divided into three groups: experimental group (transfected with HLX1 siRNAs), negative control group (transfected with negative control siRNA) and blank control group (except without siRNA, other reagents were the same as the other two groups), and transfected instantaneously.
5. DY-547-labeled negative control siRNA was transfected into HTR-8/SVneo cells. The transfection efficiency was determined by flow cytometry (FCM), and the optimum conditions for transfection efficiency were screened out.
6. Real-time fluorescence quantitative PCR (qRT-PCR) and Western Blot were used to detect the expression of HLX1 gene mRNA and protein in three groups of cells, respectively, to evaluate the interference effect.
7. Cell proliferation experiment: Three groups of HTR-8/SVneo cells were inoculated in the same amount and transfected instantaneously after overnight culture. The growth curve was drawn by MTT colorimetry every 24 hours.
8. Cell invasion assay: The ability of HTR-8/SVneo cells to penetrate the artificially reconstituted basement membrane was detected by Transwell chamber. Three groups of HTR-8/SVneo cells were inoculated in the same amount, and were transfected instantly after overnight culture. After 24 hours, the cells were suspended in serum-free medium and inoculated with Matrigel matrix gum in the same amount. In the upper compartment, 20% FCS medium was added to the lower compartment. After 24 hours of culture, the number of penetrating cells was counted by 5 visual fields randomly under 40-fold microscope.
9. Statistical methods: SPSS 11.0 software package was used for statistical analysis, and the mean (?) + standard deviation (?) + s) was used for quantitative data. One-way ANOVA was used for inter-group comparison, and P < 0.05 was statistically significant.
[results]
1.HLX1 mRNA and protein were positive in HTR-8/SVneo cells.
The transfection efficiency of 2.siRNA reached (86.3 + 2.6)%, followed by the same conditions.
3. Compared with the blank control group and the negative control group, the experimental group specifically and effectively inhibited the expression of HLX1 gene in HTR-8/SVneo cells after transfection of HLX1 siRNAs. After 48 hours of transfection, the expression of HLX1 mRNA was decreased (77.0 (+ 1.2)) (P < 0.01), and the inhibition rate of HLX1 protein was (82.6 (+ 1.2)) (P < 0.01).
4. Compared with the blank control group and the negative control group, the proliferation activity of HTR-8/SVneo cells in the experimental group decreased after transfection of HLX1 siRNAs, and the proliferation inhibition effect was the most significant 72 hours after transfection, the inhibition rate was (58.1+4.4)% (P < 0.01).
5. Compared with the blank control group and the negative control group, the invasive ability of HTR-8/SVneo cells transfected with HLX1 siRNAs was significantly inhibited (P<0.01).
[Conclusion]
HLX1 siRNAs can effectively inhibit the expression of HLX1 gene in human placental extrachorionic trophoblast HTR-8/SVneo during early pregnancy. The decrease of HLX1 gene expression can significantly inhibit the proliferation and invasiveness of human placental extrachorionic trophoblasts. The abnormal expression of HLX1 may affect the proliferation and invasiveness of trophoblasts in FGR and other pathological pregnancy. Plays an important role.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R346;R714.2
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