CAJ | 학술논문

背景：动物研究显示，自体髓核细胞移植能有效修复椎间盘退变。然而髓核细胞体外增殖能力差，这就限制了其作为种子细胞在椎间盘退变性疾病治疗中的研究及应用。 　　目的：构建包含外源性人端粒酶反转录酶基因的腺相关病毒2载体，观察其转染人髓核细胞后人端粒酶反转录酶基因的表达。 　　方法：构建pSNAV2.0-pRSV-hTERT质粒并鉴定，采用AAVMaxTM包装系统进行重组腺相关病毒2-人端粒酶反转录酶载体的构建，以 PCR 及酶切方法验证构建的质粒，构建成功后扩增，并纯化。利用腺相关病毒2-增强型绿色荧光蛋白载体转染第1代人髓核细胞，测定最佳感染复数。参照此感染复数值，确定腺相关病毒2-人端粒酶反转录酶对人髓核细胞转染的相关感染复数；对照组采用不含外源性人端粒酶反转录酶基因的腺相关病毒2进行转染。转染后1，2，4周分别采用RT-PCR对人端粒酶反转录酶基因mRNA水平进行半定量检测。 　　结果与结论：实验成功构建了腺相关病毒2-人端粒酶反转录酶载体；并获得了滴度达2×1011 v·g/mL的腺相关病毒2-人端粒酶反转录酶载体。测得腺相关病毒2-人端粒酶反转录酶载体对人髓核细胞的最佳感染复数为5×104 v·g/cel。在以1×104，5×104，1×105 v·g/cel 转染人髓核后，均可检测到人端粒酶反转录酶基因mRNA的高量表达。采用RT-PCR半定量检测方法，发现以转染后2周时人端粒酶反转录酶 mRNA表达量相对最高(P <0.05)，4周时仍可见人端粒酶反转录酶基因mRNA的稳定表达。而对照组无论在何时间点均未能检测到人端粒酶反转录酶mRNA的表达。提示利用腺相关病毒2可以成功构建包含外源性人端粒酶反转录酶基因的病毒载体，腺相关病毒2-人端粒酶反转录酶能有效转染人髓核细胞并稳定表达人端粒酶反转录酶基因mRNA，此结果可能为增强髓核细胞性能提供新的策略。揹景：動物研究顯示，自體髓覈細胞移植能有效脩複椎間盤退變。然而髓覈細胞體外增殖能力差，這就限製瞭其作為種子細胞在椎間盤退變性疾病治療中的研究及應用。 　　目的：構建包含外源性人耑粒酶反轉錄酶基因的腺相關病毒2載體，觀察其轉染人髓覈細胞後人耑粒酶反轉錄酶基因的錶達。 　　方法：構建pSNAV2.0-pRSV-hTERT質粒併鑒定，採用AAVMaxTM包裝繫統進行重組腺相關病毒2-人耑粒酶反轉錄酶載體的構建，以 PCR 及酶切方法驗證構建的質粒，構建成功後擴增，併純化。利用腺相關病毒2-增彊型綠色熒光蛋白載體轉染第1代人髓覈細胞，測定最佳感染複數。參照此感染複數值，確定腺相關病毒2-人耑粒酶反轉錄酶對人髓覈細胞轉染的相關感染複數；對照組採用不含外源性人耑粒酶反轉錄酶基因的腺相關病毒2進行轉染。轉染後1，2，4週分彆採用RT-PCR對人耑粒酶反轉錄酶基因mRNA水平進行半定量檢測。 　　結果與結論：實驗成功構建瞭腺相關病毒2-人耑粒酶反轉錄酶載體；併穫得瞭滴度達2×1011 v·g/mL的腺相關病毒2-人耑粒酶反轉錄酶載體。測得腺相關病毒2-人耑粒酶反轉錄酶載體對人髓覈細胞的最佳感染複數為5×104 v·g/cel。在以1×104，5×104，1×105 v·g/cel 轉染人髓覈後，均可檢測到人耑粒酶反轉錄酶基因mRNA的高量錶達。採用RT-PCR半定量檢測方法，髮現以轉染後2週時人耑粒酶反轉錄酶 mRNA錶達量相對最高(P <0.05)，4週時仍可見人耑粒酶反轉錄酶基因mRNA的穩定錶達。而對照組無論在何時間點均未能檢測到人耑粒酶反轉錄酶mRNA的錶達。提示利用腺相關病毒2可以成功構建包含外源性人耑粒酶反轉錄酶基因的病毒載體，腺相關病毒2-人耑粒酶反轉錄酶能有效轉染人髓覈細胞併穩定錶達人耑粒酶反轉錄酶基因mRNA，此結果可能為增彊髓覈細胞性能提供新的策略。
배경：동물연구현시，자체수핵세포이식능유효수복추간반퇴변。연이수핵세포체외증식능력차，저취한제료기작위충자세포재추간반퇴변성질병치료중적연구급응용。 　　목적：구건포함외원성인단립매반전록매기인적선상관병독2재체，관찰기전염인수핵세포후인단립매반전록매기인적표체。 　　방법：구건pSNAV2.0-pRSV-hTERT질립병감정，채용AAVMaxTM포장계통진행중조선상관병독2-인단립매반전록매재체적구건，이 PCR 급매절방법험증구건적질립，구건성공후확증，병순화。이용선상관병독2-증강형록색형광단백재체전염제1대인수핵세포，측정최가감염복수。삼조차감염복수치，학정선상관병독2-인단립매반전록매대인수핵세포전염적상관감염복수；대조조채용불함외원성인단립매반전록매기인적선상관병독2진행전염。전염후1，2，4주분별채용RT-PCR대인단립매반전록매기인mRNA수평진행반정량검측。 　　결과여결론：실험성공구건료선상관병독2-인단립매반전록매재체；병획득료적도체2×1011 v·g/mL적선상관병독2-인단립매반전록매재체。측득선상관병독2-인단립매반전록매재체대인수핵세포적최가감염복수위5×104 v·g/cel。재이1×104，5×104，1×105 v·g/cel 전염인수핵후，균가검측도인단립매반전록매기인mRNA적고량표체。채용RT-PCR반정량검측방법，발현이전염후2주시인단립매반전록매 mRNA표체량상대최고(P <0.05)，4주시잉가견인단립매반전록매기인mRNA적은정표체。이대조조무론재하시간점균미능검측도인단립매반전록매mRNA적표체。제시이용선상관병독2가이성공구건포함외원성인단립매반전록매기인적병독재체，선상관병독2-인단립매반전록매능유효전염인수핵세포병은정표체인단립매반전록매기인mRNA，차결과가능위증강수핵세포성능제공신적책략。
BACKGROUND:In animal experiments, transplantation of autologous nucleus pulposus cellscan effectively repair the intervertebral disk degeneration. However, nucleus pulposus cells have a poor ability of proliferation in vitro, which limits its application as seed cells in treatment of intervertebral disk disease. OBJECTIVE:To construct recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase and observe the human telomerase reverse transcriptase mRNA expression in human nucleus pulposus cells in vitro. METHODS:After the plasmid pSNAV2.0-pRSV-hTERT was constructed and identified, recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase were constructed, amplified and purified by AAVMaxTM package and purification system. The optimal multiplicity of infection for human nucleus pulposus cells was detected by recombinant adeno-associated virus type-2 vector carrying enhanced green fluorescent protein. According the optimal multiplicity of infection (5 × 104 v·g/cell), three different multiplicity of infection (1×104, 5×104, 1×105 v·g/cell) of recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase were determined to transfect the first passage human nucleus pulposus cells in vitro. In control group, the cells were transfected with adeno-associated virus type-2 vector without human telomerase reverse transcriptase. At 1, 2, 4 weeks after transfection, mRNA expression of human telomerase reverse transcriptase in human nucleus pulposus cells were semi-quantitatively detected by RT-PCR. RESULTS AND CONCLUSION:The recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase was successful y constructed, and the titer of the obtained vector was more than 2×1011 v·g/mL. The optimal multiplicity of infection was 5×104 v·g/cell. The mRNA expression of human telomerase reverse transcriptase in human nucleus pulposus cells could be detected in different multiplicity of infection (1×104, 5×104, 1×105 v·g/cell). At 2 weeks post-transfection, mRNA expression of human nucleus pulposus cells was the highest (P<0.05), as detected by semi-quantitative RT-PCR. Moreover, the stable and high mRNA expression of human telomerase reverse transcriptase could be detected at 4 weeks post-transfection. In control group, no human telomerase reverse transcriptase mRNA expression was found. The recombinant adeno-associated virus type-2 vector carrying human telomerase reverse transcriptase can be successful y constructed, and can mediate a stable mRNA expression of human telomerase reverse transcriptase in human nucleus pulposus cells. Our findings provide a novel strategy of enhancing the properties of nucleus pulposus cells.