| Periodontal disease results in the loss of the periodontium, which includes cementum, periodontal ligament and bone, so it is difficult to reconstitute the injured tissues of the periodontal attachment apparatus physiologically and functionally. With deeply development of tissue engineering and gene therapy recently,some experiments have indicated that the tansfected gingival fibroblast can obtain new biological characteristics and stably express recombinant protein for tissue in vivo. The tansfected gingival fibroblast may be good seed cell for periodontal tissue engineering.We combine gene transfection with tissue engineering. Human transforming growth factor P-1 (TGF-β1) gene was transfected into human gingival fibroblast(GF).In the condition that normal culture and existing of putative periodontopathic factor lipopolysaccharide (LPS), the biological character and the expression of TGF-P receptor and Smad2/3 protein have been observed. This study tries to realize TGF- β1 Signal transduction pathway in humnan GF transfected by TGF-β1 gene and to estimate the potential effects of it as seed cell for periodontal tissue engineering. Several experiments as below are carried on in this dissertation: 1. The expression vector for TGF-β1 was amplified in E.coli DH5 α ,then the positive recombinants were extracted, purified, and identified by restriction endonuclease digestion. TGF-β1 gene was transfected into thecultured human GF by using lipofectamine. Positive clones were selected with G-418. The expression of TGF-β1 in transfected GF were determined by immunohistochemistry and Indirect ELISA. Results: The two fragments digested from pcDNA3.0-hTGF-β1 by Xhol I and Xba I represented 5.4kb and 1.2kb by agaros electrophoresis; Transfected GF showed strong positive activity. Expression of TGF-pi lasted for more than ten generation. Image analysis indicated the expression of TGF-pi in the transfected GF was higher than the control significantly(P<0.01). Indirect ELISA showed that transfected human GF could produce TGF-pi in a steady level of 1ng/mL. Conclusion: Transfected GF could effectively express active TGF-β1 and produce biological efficiency.2. The effects of transfection with TGF- β1 gene on the biological behaviours of human GF and the characteristics of the culture supernatant fluid of transfected human GF were investigated with Enzyme kinetics and MTT chromometry method. Results: The shape of parts of TGF- β 1 gene transfected GF changed from fusiform to polygonal.Compared the culture supernatant fluid of transfected human GF with which without transfection, the former fluid evidently promoted the proliferation of human PDLCs and GF(P<0.01), and made ALP activity of them significantly improved, the promotion is in a time-dependent way. ALP activity of transfected human GF is also much higher than the control group, and very close to ALP activity of human PDLCs. Conclusion: GF transfected by TGF- β 1 gene not only can induce themselves proliferation and differentiation toward osteoblast-like cells, but also through the paracrine way induce proliferation and differentiation of human PDLCs and GF.3. The expression of type I and II TGF- β receptors and Smad2/3 proteins oftransfected human GF and GF without transfection were determined by Immunohistochemistry and cellular immunofluorescence techniques. Results: The expression of type I and II TGF- β receptor of transfectedGF is higher than that of GF without transfection.The positive signal in cytoplasm of transfected GF is stronger than that of GF without transfection (P<0.05). Smad2/3 proteins of GF without transfection are mostly expressed in cytoplasm. On the contrary, Smad2/3 proteins of transfected GF and GF which cultured with the supernatant fluid of transfected GF are mostly expressed in nucleus.The results clues on that the Smad2/3 complex, which has been activated by TGF- β receptors,translocates from the cytoplasm into the nuclear.There was the same result detected by cellular immuno- fluorescence. Smad2/3-associated fluorescence in nuclear of transfected GF shows a low degree compared with that of GF without transfection and GF which cultured with the supernatant fluid of transfected GF. The results show that GF can down-regulated the expression of Smad2,3 when the moderate reaction caused by TGF- β 1 have been done. Conclusion:GF is one of the target cells of TGF- β . The results proposed that Smad2,3 was the specific downstream signal transducer for TGF- β 1, the Smad2,3 complex can translocates from the cytoplasm into the nuclear when they are activated by TGF- β receptors.The cells of tissue have a good ability of regulating the responsion to the stimulating by TGF- β .They can make a moderate reaction without any pathological change.4. MTT and RIA (radio immunoassay) was used to assess the biologicalcharacter of GF transfected with the TGF- β 1 gene and GF without transfection after they were stimulated by LPS. Results: The study indicated that LPS had affected proliferation of transfected GF and GF without transfection at all concentrations examined. The study showed that the growth inhibitory effects were found only when considerably higher concentrations of LPS were given,such as 10μg/mL, 100μg/mL. In contrast, treatment with low concentrations of LPS exhibited the enchancement of proliferation,such as 0.01 μg/mL, 0.1μg/mL. Therefore, the growth inhibitory effects of LPS are mainly relevent to the...
|
PDF Full Text Request