| Telomere is a special structure of eukaryotes linear chromosomes ends which is composed ofhighly conserved non-coding DNA simple repeat sequences and telomeric binding proteins. Itplayed an important role in maintaining the integrity of chromosomes. Telomere progressivelyshortened in dividing cells. if telomere haven’t extend effectively, it will reach at its limit lengthand lead to cell aging and apoptosis. Telomerase could take G-overhang of telomere3’ end asprimer, its own RNA subunit as template; add synthetic telomeric repeat sequence to telomereend to prolong telomere by the catalysis of telomerase reverse transcriptase subunit. Telomeraseactivity rarely expressed in normal somatic cells, but highly expressed in tumor cells and celllines. This means that telomerase is close related with cancer, and it could be a diagnostic markerof early human cancer. At present, there are many tumor therapies focus on telomere andtelomerase, but these methods needs improve which were imperfect. Telomerase RNA templatesequence determined the specificity of synthetic telomeric repeat sequence, thus telomerase RNAtemplate site-mutants may have significant impacts on telomerase activity, telomere length ortumor cell growth. This paper intended to reveal the effect of hTER template site-mutants onhuman mammary cancer cell lines Bcap-37by detecting the change of telomerase activity,telomere length and apoptosis.This paper took tumor of nude mice which inoculated by human mammary cancer cellBcap-37as the experimental material, they divided into MT-hTER group and control group. FirstI extracted telomerase from MT-hTER group samples and control group samples, and detectedthe protein content of the extract. Then run the enzymatic reaction, telomerase will reversetranscribing telomeric DNA sequence base on the leading primer. Normal group and negativecontrol group have set. Negative control group’s telomerase activity has inactivated beforerunning the reaction. End the enzymatic reaction, and detected the PCR products ssDNA contentby NanoDrop2000Spectrophotometer. Telomerase activity could be indicated by the differentialof ssDNA content between normal group and negative control group. We can obtain the Effect ofhTER template site-mutants on human mammary cancer lines cells Bcap-37telomerase bycontrasting telomerase activity between MT-hTER group and control group. Contrast resultsshowed that telomerase activity of MT-hTER group samples were weaker than control group. This indicates that hTER template site-mutants may partly changed telomerase activity of humanmammary cancer cell Bcap-37.Genome DNA was extracted from MT-hTER group samples and control group samples;make it jointed with the special adaptor by T4DNA ligase. Base on human genome DNAsubtelomeric sequence from NCBI gene bank database, the upstream primer which couldpeculiarly amplify special telomere have been designed. I amplified special telomere of humanmammary cancer cell Bcap-37genome DNA by PCR. I analyzed PCR products by agarose gelelectrophoresis and GeneTools software, I got PCR products content and the molecular weight of1/2PCR products content, calculated the mean length of telomere. The measurement resultsshowed that the mean length of MT-hTER group chromosome telomeres were shorter thancontrol group. This indicates that hTER template site-mutants may partly changed the meanlength of human mammary cancer cell Bcap-37telomere.The paraffin sections of human mammary cancer cell Bcap-37have stained by feulgenstaining method. We could observe the claret-colored cell nucleus through the optical microscope.The observation results showed that the cell nucleuses of MT-hTER group were unstable, sizeswere different, nuclear condensation appeared frequently. But the cell nucleuses of control groupwere stable, sizes were mostly same, nuclear condensation appeared rarely. This indicated thathTER template site-mutants may partly induced apoptosis in the proliferation of humanmammary cancer cell lines Bcap-37. |
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