大鼠肌肉挫傷后肌鈣蛋白I mRNA表達與損傷時間關系的研究
發(fā)布時間：2018-09-07 14:23
【摘要】： 目的:應用實時熒光定量PCR技術(shù)檢測大鼠肌肉挫傷后骨骼肌肌鈣蛋白I(skeletal troponin I,sTnI)mRNA表達,探尋其時序性表達規(guī)律,探討sTnI mRNA表達變化應用于損傷時間推斷的可行性,旨在為法醫(yī)學早期損傷時間的推斷提供科學依據(jù)。 方法:選取健康成年雄性SD大鼠63只,隨機分為正常對照組(6只)、死后損傷組(9只)和生前損傷組(48只)。均實施麻醉、褪毛后,利用改進的自由落體裝置,250g重力錘自150cm高度垂直自由落下,造成大鼠右后肢大腿內(nèi)側(cè)肌群挫傷。損傷后0.5h、1h、6h、12h、18h、24h、30h、36h八個時間點(每個時間點6只)取挫傷處肌肉組織50mg,置于液氮中保存?zhèn)溆?正常對照組大鼠未打擊造傷;死后損傷組脫頸處死后,自由落體打擊同一部位,其余處理與實驗組相同。以膜吸附技術(shù)為基礎的SV Total RNA Isolation System提取肌肉組織中的總RNA,逆轉(zhuǎn)錄合成cDNA第一條鏈,梯度濃度稀釋cDNA原液分別制作sTnI和參比基因的相對定量標準曲線;選取管家基因核糖體蛋白L32(Ribosomal Protein L32,RPL32)為參比基因,利用SYBR Green I嵌合熒光法實時定量PCR檢測sTnI mRNA逆轉(zhuǎn)錄合成cDNA的相對表達量,分別與損傷時間進行統(tǒng)計學分析。結(jié)果:(1)Agilent 2100芯片生物分析儀和2%甲醛變性瓊脂糖凝膠電泳檢測總RNA純度、濃度及完整性較好,均可滿足下一步實驗的要求。 (2)sTnI與參比基因RPL32擴增效率分別為103.6%和100.5%,一致性較好,說明參比基因選擇正確;擴增曲線拐點清楚,基線平而無上揚現(xiàn)象,說明引物設計特異性強、反應性能良好;sTnI基因融解溫度為88°C,RPL32基因融解溫度為84°C,兩者融解曲線均為單一峰型,基底窄而峰高,表明并無引物二聚體等非特異性擴增產(chǎn)生。 (3)大鼠骨骼肌挫傷后sTnI mRNA表達明顯下調(diào)(P0.05),挫傷后0.5h、1h、6h sTnI mRNA表達量分別為正常組的78.17%、41.58%、32.13%且差異具有統(tǒng)計學意義(P0.05),6h~36h sTnI mRNA表達量與對照組相比差異無統(tǒng)計學意義(P0.05)。 (4)為了比較生前損傷組正常肌肉與損傷肌肉中sTnI mRNA的表達水平,選取挫傷后18h組大鼠左后肢同一部位取材。結(jié)果表明正常肌肉中sTnI mRNA表達與正常對照組相比無差異(P0.05),而同一個體損傷肌肉中sTnI mRNA表達與正常肌肉中sTnI mRNA表達存在差異(P0.05)。 (5)與正常對照組相比,死后損傷組雙后肢sTnI mRNA的量約下降了30%(P0.05),死后損傷組各時間點sTnI mRNA的量無差異(P0.05)。 結(jié)論:(1)生前損傷組正常肌肉中sTnI mRNA表達與正常對照組相比無差異(P0.05),而同一個體損傷肌肉中sTnI mRNA表達與正常肌肉中sTnI mRNA表達存在差異(P0.05), sTnI mRNA的表達僅在損傷肌肉中發(fā)生下調(diào),同一個體未遭受損傷部位的肌肉可以作為評價損傷肌肉的對照。 (2)大鼠肌肉挫傷后36h內(nèi)sTnI mRNA呈時序性表達下調(diào)趨勢,與損傷時間有一定的關系,其時序性變化可望作為骨骼肌早期損傷時間推斷的指標之一,服務于法醫(yī)學實踐。 (3)實時熒光定量PCR技術(shù)檢測分子水平的變化敏感而且準確,適合法醫(yī)學研究及檢案的需要。
[Abstract]:AIM: To detect the expression of skeletal troponin I (sTnI) mRNA in skeletal muscle of rats after muscle contusion by real-time fluorescence quantitative PCR, and to explore the regularity of its sequential expression, and to explore the feasibility of applying the changes of sTnI mRNA expression to the estimation of injury time, so as to provide scientific basis for forensic early injury time estimation.
Methods: 63 healthy adult male SD rats were randomly divided into normal control group (6 rats), postmortem injury group (9 rats) and prenatal injury group (48 rats). Fifty mg of muscle tissue was taken from the contusion site at eight time points (six at each time point) at 2h, 18h, 24h, 30h and 36h, and stored in liquid nitrogen for reserve; rats in the normal control group were not injured by percussion; rats in the postmortem injury group were executed after neck removal by free fall, and the rest of the treatments were the same as those in the experimental group. Total RNA was extracted from muscle tissues and the first strand of the cDNA was synthesized by reverse transcription. The relative quantitative standard curves of sTnI and reference genes were made by gradient dilution of the original cDNA. The housekeeping gene ribosomal protein L32 (RPL32) was selected as the reference gene and the sTnI mRNA inversion was detected by SYBR Green I chimeric fluorescence real-time quantitative PCR. Results: (1) Agilent 2100 microarray Bioanalyzer and 2% formaldehyde denatured agarose gel electrophoresis were able to detect the purity, concentration and integrity of total RNA, which could meet the requirements of the next experiment.
(2) The amplification efficiencies of sTnI and RPL32 were 103.6% and 100.5% respectively, which indicated that the selection of reference genes was correct; the inflection point of amplification curve was clear and the baseline was flat without rising phenomenon, indicating that the primer design was specific and the reaction performance was good; the melting temperature of sTnI gene was 88 C, and the melting temperature of RPL32 gene was 84 C. For a single peak type, the basal and narrow peaks showed no primer and no specific amplification of primers such as two dimers.
(3) The expression of sTnI mRNA was significantly down-regulated after skeletal muscle contusion in rats (P 0.05). The expression of sTnI mRNA was 78.17%, 41.58%, 32.13% in the normal group at 0.5 h, 1 h and 6 h after contusion, and the difference was statistically significant (P 0.05).
(4) In order to compare the expression of sTnI mRNA in normal and injured muscles of prenatal injury group, the left hind limbs of rats in 18 h post-contusion group were taken from the same site. The results showed that there was no difference in the expression of sTnI mRNA between normal muscle and normal control group (P 0.05), but the expression of sTnI mRNA in injured muscles of the same body was higher than that in normal muscle (P 0.05). There were differences (P0.05).
(5) Compared with the normal control group, the amount of sTnI mRNA in both hind limbs of postmortem injury group decreased by about 30% (P 0.05), and there was no difference in the amount of sTnI mRNA at each time point of postmortem injury group (P 0.05).
Conclusion: (1) The expression of sTnI mRNA in normal muscle of prenatal injury group was not different from that of normal control group (P 0.05), but the expression of sTnI mRNA in injured muscle of the same body was different from that in normal muscle (P 0.05). The expression of sTnI mRNA was down-regulated only in injured muscle, and the muscles of the same body could act as injured muscle. To evaluate the control of injured muscles.
(2) The expression of sTnI mRNA was down-regulated sequentially within 36 hours after muscle contusion in rats, which was related to the time of injury. The time-series changes of sTnI mRNA may serve as one of the indicators for estimating the time of early skeletal muscle injury and serve forensic practice.
(3) Real-time fluorescence quantitative PCR is sensitive and accurate in detecting the changes of molecular level, which is suitable for forensic research and case detection.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：D919
本文編號：2228519
[Abstract]:AIM: To detect the expression of skeletal troponin I (sTnI) mRNA in skeletal muscle of rats after muscle contusion by real-time fluorescence quantitative PCR, and to explore the regularity of its sequential expression, and to explore the feasibility of applying the changes of sTnI mRNA expression to the estimation of injury time, so as to provide scientific basis for forensic early injury time estimation.
Methods: 63 healthy adult male SD rats were randomly divided into normal control group (6 rats), postmortem injury group (9 rats) and prenatal injury group (48 rats). Fifty mg of muscle tissue was taken from the contusion site at eight time points (six at each time point) at 2h, 18h, 24h, 30h and 36h, and stored in liquid nitrogen for reserve; rats in the normal control group were not injured by percussion; rats in the postmortem injury group were executed after neck removal by free fall, and the rest of the treatments were the same as those in the experimental group. Total RNA was extracted from muscle tissues and the first strand of the cDNA was synthesized by reverse transcription. The relative quantitative standard curves of sTnI and reference genes were made by gradient dilution of the original cDNA. The housekeeping gene ribosomal protein L32 (RPL32) was selected as the reference gene and the sTnI mRNA inversion was detected by SYBR Green I chimeric fluorescence real-time quantitative PCR. Results: (1) Agilent 2100 microarray Bioanalyzer and 2% formaldehyde denatured agarose gel electrophoresis were able to detect the purity, concentration and integrity of total RNA, which could meet the requirements of the next experiment.
(2) The amplification efficiencies of sTnI and RPL32 were 103.6% and 100.5% respectively, which indicated that the selection of reference genes was correct; the inflection point of amplification curve was clear and the baseline was flat without rising phenomenon, indicating that the primer design was specific and the reaction performance was good; the melting temperature of sTnI gene was 88 C, and the melting temperature of RPL32 gene was 84 C. For a single peak type, the basal and narrow peaks showed no primer and no specific amplification of primers such as two dimers.
(3) The expression of sTnI mRNA was significantly down-regulated after skeletal muscle contusion in rats (P 0.05). The expression of sTnI mRNA was 78.17%, 41.58%, 32.13% in the normal group at 0.5 h, 1 h and 6 h after contusion, and the difference was statistically significant (P 0.05).
(4) In order to compare the expression of sTnI mRNA in normal and injured muscles of prenatal injury group, the left hind limbs of rats in 18 h post-contusion group were taken from the same site. The results showed that there was no difference in the expression of sTnI mRNA between normal muscle and normal control group (P 0.05), but the expression of sTnI mRNA in injured muscles of the same body was higher than that in normal muscle (P 0.05). There were differences (P0.05).
(5) Compared with the normal control group, the amount of sTnI mRNA in both hind limbs of postmortem injury group decreased by about 30% (P 0.05), and there was no difference in the amount of sTnI mRNA at each time point of postmortem injury group (P 0.05).
Conclusion: (1) The expression of sTnI mRNA in normal muscle of prenatal injury group was not different from that of normal control group (P 0.05), but the expression of sTnI mRNA in injured muscle of the same body was different from that in normal muscle (P 0.05). The expression of sTnI mRNA was down-regulated only in injured muscle, and the muscles of the same body could act as injured muscle. To evaluate the control of injured muscles.
(2) The expression of sTnI mRNA was down-regulated sequentially within 36 hours after muscle contusion in rats, which was related to the time of injury. The time-series changes of sTnI mRNA may serve as one of the indicators for estimating the time of early skeletal muscle injury and serve forensic practice.
(3) Real-time fluorescence quantitative PCR is sensitive and accurate in detecting the changes of molecular level, which is suitable for forensic research and case detection.
【學位授予單位】：山西醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：D919
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，本文編號：2228519
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