Study On The Construction Of HTR Relative Expression In Cells, And Study On The Detection Of Telomerase Activity

Telomerase is a ribonucleoprotein complex (RNP) composed by its RNA component and protein subunits.Telomerase can synthesize telomeric DNA onto chromosomal ends using its own RNA component as a template, elongate the length of telomere, increase cell life and even induce cell immortalization. In most normal tissues, telomerase activity can not be detected or nearly be detected. However, telomerase plays an important role in tumorigenesis and development of malignant tumor, which makes it a possible new target in gene therapy.This thesis mainly focuses on: (1) Effects of the expression of telomerase antisense RNA gene on the morphology and biochemical characters of human liver cancer SMMC-7721 cells. (2) Effects of the expression of telomerase RNA gene on the telomerase activity in human normal peripheral blood leukocytes, cell growth and cell proliferation. (3) Establishing a new method which can quantitatively determinate telomerase activity.1. Human telomerase RNA component (hTR)gene was cloned from HeLa cells using gene engineering technology.The hTR gene was then reverse inverted into retrovirus vector pLNCX and constructed an antisense recombinant plasmid pLNCX-aTR. The obtained recombinant-5-hTRplasmid was tranfected into human liver cancer SMMC-7721 cells. All data suggested the expression of pLNCX-aTR could condense cell nucleus and increase nuclear fluorescence intensity, effectively repress the telomerase activity, cell growth and cell proliferation, and induce cell apoptosis. DNA ladder bands with a periodicity of about 200 bp were clearly seen in agarose gel electrophoresis pattern. These results comply with cell morphology and biochemical characters of apoptotic cells. We concluded that expression of antisense hTR gene could result in the apoptosis of SMMC-7721 cells. Gene therapy of tumor using telomerase as a target is a prospective new approach with tremendous potentials.2. Transfection of telomerase positive sense expression plasmid pLNCX-TR into human normal peripheral leukocytes didn't necessarily change the level of telomerase activity in cells, It indicated that expression of exogenous hTR gene could not activate the expression of hTERT and increase telomerase activity. Flow cytometry (FCM) analysis showed that expression of exogenous hTR gene repressed the proliferation of leukocytes and resulted in cell apoptosis. We concluded that excessive expression of exogenous hTR gene may compete with the endogenous telomerase RNA and prevent RNA template from combining with telomeric DNA, thus repressing the elongation of telomeric DNA(telomeres) and causing cell aging and cell death.-6-3. Some modifications have been made to overcome the limitation of conventional telomeric repeat amplification protocol (TRAP) assay. That is to add a special fluorescence-based DNA internal standard in the telomerase elongated TS primers, then do PCR amplification after a step of refinement(hydroxybenzene/chloroform extracting, and deposited by ethanol). Sequencing analysis of PCR product was done on 310 Gene Scan Analysis?.1.2 DNA Sequencer to determine telomerase activity. Notably, this method eliminated the restraining factors of Taq DNA polymerase, making it possible to erase the sample differences met in PCR and eradicate the annoying phenomena of pseudo negative results. Comparison of telomerase activity in a few cell lines showed that application of DNA sequencer in detecting telomerase enzyme activity is applicable in quantitative and qualitative determination of telomerase activity. The result of this method could be available within 4-5 hours with high sensitivity and less handling time. It provided a reliable means to study on regulative mechanism of telomerase activity and relationship between telomerase activation and malignancy. It is also a novel and reliable method in clinical diagnostics and therapeutics of tumor malignancy.