RNAi技術(shù)抑制同源異型盒基因HLX1表達(dá)對滋養(yǎng)細(xì)胞增殖和侵襲的影響
發(fā)布時間：2018-08-16 09:04
【摘要】： 【研究背景及目的】 同源異型盒基因是一類重要的調(diào)控細(xì)胞分化和形態(tài)發(fā)育的轉(zhuǎn)錄因子基因家族,其中HLX1基因與胚胎來源細(xì)胞和造血系細(xì)胞的增殖、分化行為密切相關(guān)。近年來研究陸續(xù)表明,HLX1基因?qū)τ谡ＬケP形成起重要作用;HLX1蛋白在細(xì)胞滋養(yǎng)細(xì)胞來源的細(xì)胞系HTR-8/SVneo、SGHPL-4、JEG-3、JAR和BeWo的細(xì)胞中均有表達(dá);病理妊娠如特發(fā)性胎兒宮內(nèi)生長受限(FGR)的胎盤中HLX1基因表達(dá)水平明顯降低。本研究旨在運用RNAi技術(shù)抑制滋養(yǎng)細(xì)胞株HTR-8/SVneo中HLX1基因的表達(dá),探討其對體外滋養(yǎng)細(xì)胞增殖和侵襲行為的影響,為HLX1基因異常表達(dá)可能參與FGR等病理妊娠的發(fā)病提供基礎(chǔ)理論支持。 【方法】 1.復(fù)蘇、培養(yǎng)人早孕期胎盤絨毛膜外滋養(yǎng)細(xì)胞株HTR-8/SVneo至對數(shù)生長期備用。 2.應(yīng)用反轉(zhuǎn)錄-聚合酶鏈反應(yīng)(RT-PCR)、Western Blot方法檢測HTR-8/SVneo細(xì)胞中HLX1 mRNA和蛋白質(zhì)的表達(dá)。 3.依據(jù)GeneBank中HLX1 cDNA序列號NM_021958,設(shè)計并化學(xué)合成siRNA序列,分別為:4條特異性HLX1 siRNA的預(yù)混試劑;1條非特異性陰性對照siRNA試劑,DY-547熒光標(biāo)記。 4.HTR-8/SVneo細(xì)胞常規(guī)體外培養(yǎng),設(shè)為三組:實驗組(轉(zhuǎn)染HLX1 siRNAs)、陰性對照組(轉(zhuǎn)染陰性對照siRNA)以及空白對照組(除不含siRNA,余試劑與另兩組均相同),分別進(jìn)行瞬時轉(zhuǎn)染。 5.將DY-547標(biāo)記的陰性對照siRNA轉(zhuǎn)染入HTR-8/SVneo細(xì)胞,應(yīng)用流式細(xì)胞術(shù)(FCM)測定轉(zhuǎn)染效率,篩選出轉(zhuǎn)染效率最佳優(yōu)化條件進(jìn)行后續(xù)實驗。 6.應(yīng)用實時熒光定量PCR(qRT-PCR)和Western Blot方法分別檢測三組細(xì)胞轉(zhuǎn)染后HLX1基因mRNA和蛋白的表達(dá),評價干擾效果。 7.細(xì)胞增殖實驗:將三組HTR-8/SVneo細(xì)胞以相同數(shù)量接種,過夜培養(yǎng)后進(jìn)行瞬時轉(zhuǎn)染,應(yīng)用四甲基偶氮唑鹽(MTT)比色法每隔24測定吸光度值后繪制生長曲線。 8.細(xì)胞侵襲實驗:應(yīng)用Transwell小室檢測轉(zhuǎn)染后各組HTR-8/SVneo細(xì)胞穿透人工重組基底膜的能力。三組HTR-8/SVneo細(xì)胞以相同數(shù)量接種,過夜培養(yǎng)后進(jìn)行瞬時轉(zhuǎn)染,24小時后,用無血清培養(yǎng)液重懸細(xì)胞并以相同數(shù)量接種于包被有Matrigel基質(zhì)膠的小室上室,下室加入20%FCS的培養(yǎng)液。繼續(xù)培養(yǎng)24h,40倍鏡下隨機(jī)取5個視野計數(shù)穿膜細(xì)胞數(shù)。 9.統(tǒng)計學(xué)方法:應(yīng)用SPSS 11.0軟件包進(jìn)行統(tǒng)計分析,以均數(shù)±標(biāo)準(zhǔn)差((?)±s)表示定量資料數(shù)據(jù),組間比較采用單因素方差分析(ANOVA),認(rèn)為P＜0.05有統(tǒng)計學(xué)意義。 【結(jié)果】 1.HLX1 mRNA和蛋白在HTR-8/SVneo細(xì)胞中均呈陽性表達(dá)。 2.siRNA轉(zhuǎn)染效率優(yōu)化后可達(dá)(86.3±2.6)%,以相同條件進(jìn)行后續(xù)實驗。 3.與空白對照組及陰性對照組相比,實驗組轉(zhuǎn)染HLX1 siRNAs后特異且有效地抑制了HTR-8/SVneo細(xì)胞中HLX1基因的表達(dá),轉(zhuǎn)染48h后HLX1 mRNA的表達(dá)降低(77.0±1.2)%(P＜0.01),轉(zhuǎn)染72h后HLX1蛋白的抑制率為(82.6±1.2)%(P＜0.01)。 4.與空白對照組及陰性對照組相比,實驗組轉(zhuǎn)染HLX1 siRNAs后HTR-8/SVneo細(xì)胞的增殖活性下降,轉(zhuǎn)染72h后增殖抑制效應(yīng)最為顯著,抑制率為(58.1±4.4)%(P＜0.01)。 5.與空白對照組及陰性對照組相比,實驗組轉(zhuǎn)染HLX1 siRNAs后HTR-8/SVneo細(xì)胞穿過Matrigel基質(zhì)膠的侵襲能力受到顯著抑制,差異有統(tǒng)計學(xué)意義(P＜0.01)。 【結(jié)論】 HLX1 siRNAs可有效抑制人早孕期胎盤絨毛膜外滋養(yǎng)細(xì)胞HTR-8/SVneo中HLX1基因的表達(dá),HLX1基因表達(dá)下降可以顯著抑制滋養(yǎng)細(xì)胞的增殖活性和侵襲能力,它的表達(dá)異常可能通過影響滋養(yǎng)細(xì)胞增殖、侵襲等生物學(xué)性能而在FGR等病理妊娠的發(fā)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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2009
【分類號】：R346;R714.2
【參考文獻(xiàn)】
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