CAJ | 학술논문

目的通过构建真核表达pEGFP-C2-ASIC2a质粒,转染大鼠关节软骨细胞,建立ASIC2a在关节软骨细胞中过表达的模型,观察 ASIC2a mRNA及其蛋白在细胞中的表达。方法从大鼠脑组织中提取目的基因ASIC2a,并用EcoR玉和Kpn玉进行双酶切,同时用这两种酶双酶切质粒pEGFP-C2,将其酶切产物按常规方法连接并转化入大肠杆菌DH5a,挑单克隆菌进行培养,提取质粒,再通过双酶切鉴定及测序后,用Lipofectamine 2000将所构建质粒转染入关节软骨细胞,在荧光显微镜下观察绿色荧光蛋白的表达,使用RT-PCR和Western blot法检测ASIC2a mRNA和蛋白表达,以鉴定模型建立成功与否。结果进行双酶切鉴定目的条带清晰准确,转染后可在荧光显微镜下观察到绿色荧光表达,通过RT-PCR可发现mRNA转录,Western blot法可发现目的蛋白表达。结论成功构建重组pEGFP-C2-ASIC2a表达载体,将用于进一步观察ASICs对软骨细胞的影响。
목적통과구건진핵표체pEGFP-C2-ASIC2a질립,전염대서관절연골세포,건립ASIC2a재관절연골세포중과표체적모형,관찰 ASIC2a mRNA급기단백재세포중적표체。방법종대서뇌조직중제취목적기인ASIC2a,병용EcoR옥화Kpn옥진행쌍매절,동시용저량충매쌍매절질립pEGFP-C2,장기매절산물안상규방법련접병전화입대장간균DH5a,도단극륭균진행배양,제취질립,재통과쌍매절감정급측서후,용Lipofectamine 2000장소구건질립전염입관절연골세포,재형광현미경하관찰록색형광단백적표체,사용RT-PCR화Western blot법검측ASIC2a mRNA화단백표체,이감정모형건립성공여부。결과진행쌍매절감정목적조대청석준학,전염후가재형광현미경하관찰도록색형광표체,통과RT-PCR가발현mRNA전록,Western blot법가발현목적단백표체。결론성공구건중조pEGFP-C2-ASIC2a표체재체,장용우진일보관찰ASICs대연골세포적영향。
Objective To construct eukaryotic expression vector of acid sensing ion channel-2 a ( ASIC2 a ) , and transfect it into the articular cartilage cells of rats to make the model of overexpression of ASIC2a. Methods Got the cDNA of ASIC2a from rat brain, then ASIC2a cDNA was amplified by PCR and cut with double enzyme EcoR I and Kpn I, then inserted into the eukaryotic expression vetor pEGFP-C2. The recombinant vector was verified by PCR, restriction enzymes cut and sequencing identified. We used Lipofectamine 2000 transfection reagent to trans-fect the plasmid into the cartilage cells of rats, then observed GFP expression under the fluorescence microscope. We also determined the relative expression of the mRNA and protein of ASIC2 a by RT-PCR and Western blot to i-dentify whether the model of overexpression was constructed successfully. Results The plasmid was identified by enzyme cutting where the purpose gene was included, and the electrophoretic stripes were accurate and distinct, al-so GFP could be detected in the transfected the articular cartilage cells of rats. ASIC2a gene expression could be detected by PCR, also its protein expression was detected by Western blot. Conclusion The model of overexpres-sion is constructed successfully which can be used to observe the effect on cartilage cells of the acid sensing ion channels expression.