Establisment Of In Vitro Model Of Bovine Mammary Gland Epithelial Cells In Vitro Model By Adenovirus-meditated Human Telomerase Reverse Transcriptase (Htert) Gene

Mammary gland epithelial cells are target cells which have been used to establish mammary bioreactor and valuate mammary gland-specific expression-vector. At present, there are not enough cell lines to be used to detect mammary gland-specific expression-vector, because mammary gland epithelial cells (MGEs) was very difficult to culture in vitro. Furthermore, many research results have showed that these cells appear to be suitable for breast-related diseases research because of genomic aberration, cellular heterogeneity and malignancy. Therefore, it is necessary to establish a valid method to be used as in vitro models for the study of the mammary gland epithelial cells. In the present study, the culture conditions were optimized, and an adenovirus vector which carried hTERT was constructed and transfered into bMGEs. Characteristics and function of the positive cells (hTERT-bMGEs) were detected. The results are as following:1. The twice tissue culture method was the best method to obtain bMGEs which were absolutely purified, having stable characteristics, and vigorous proliferation.2. The results of comparing study of seceral different culture media of bMGEs, showed that the media which as consist of 1:1 Dulbecco's Modified Eagle's Medium/F12 (DMEM/F12, Invitrogen), supplied with 10% fetal calf serum (FCS), 2 mM L-glutamine, 5μg/mL Hydrocortisone, 10ng/mL epidermal growth factor (EGF), 10μL/mL Insulin-Transferrin-Selenium-A Supplement (100X) (ITS), and 5μg/mL insulin was the best media for bMGEs culture.3. pEGFP-hTERT as a fusion gene, was constructed and transfered into bMGEs. In order to selecting the best strategy for high-efficiency transfection, the conditions of transfer were optimized and detected by fluorescence microscopy. The results showed that the growth status of bMGEs played an important role in reansfering.4. An adenovirus vector which carrying hTERT gene was constructed and transfered into bMGEs. The initial titer was 3×10⁷ pfu/mL, and after amplification it was 2.3×10¹⁰ pfu/mL. The MOI was 10-20 infection bMGEs.5. The adenovirus- and plasmid-mediated hTERT were compared in terms of the positive cloning and transgenic efficiency. The infection efficiency of adenovirus group was high up to 76±1%, and the average transfection efficiency of plasmid groups was 33±2%; the positive cloning efficeiency of adenovirus group was 50%, but that of plasmid groups was zero. The results indicated that the adenovirus group had higher infection efficiency and produced a positive polyclone population.6. hTERT-bMGEs were analized by flow cytometry, immunofluorescence, Real-time quantitative PCR, western blot, proliferation assays, and TRAP-PCR, which showed that the adenovirus-mediated hTERT gene could not only extend the cell lifespan, but also prolong the expression of hTERT gene on bMGEs. Expression of hTERT gene in bMGEs cells was maintained only passge fourteen, while the positive polyclone population hTERT-bMGEs could grow more than passage forty.7. The result of Southen blot analysis showed that the average TRF of hTERT-bMGEs at passage 5, 12, 18 and 27 was 17.73 kb±0.80 kb, and the TRF of bMGEs at passage 4 was 16.43 kb, while the TRF of bMGEs at passage 13 was 13.4 kb. The results showed that hTERT could extend bovine mammary cell lifetime at early span by maintaining telemeter length. At late span, expressions of some tumor suppressor genes and transcription factor were found to down-regulate in hTERT-bMGEs, demdemonstrating that the change of p21WAF1-p53 signal path might paly a role in cell proliferation and lifetime maintainance.8. The mammary gland-specific expression-vector carring human lysozyme had been transfered into hTERT-bMGEs. The results showed that hTERT-bMGEs could produce high transfection efficiency and polyclone efficiency rate, and human lysozyme could express in hTERT-bMGEs under hormone pressure.9. The bMGEs at passge 10 and the hTERT-bMGEs at passge 18 and 28 were used as donor cells for analysis the development of nuclear transferred (NT) embryoes, the results demonstrate that hTERT-bMGEs at passge 18 could improve the NT-embryoes development and quality, comparing to bMGEs at passge 10, and which domenstrated that the hTERT-bMGEs could be used for NT.