[Construction of human Bcl-6 3'UTR reporter vector and expression vector and their functional assessment].

OBJECTIVE: To observe the direct regulation of miR-127 on Bcl-6 and the effect of Bcl-6 in rescuing miR-127-induced cell cycle and cell growth inhibition. METHODS: The 3'UTR and coding region of human bcl-6 gene were amplified by PCR and cloned into pcDNA3.0-Luc and pcDNA3.0-Flag vectors, respectively. Mutations were introduced into the seed sequences of the predicted miR-127 target sites within the Bcl-6 3'UTR using recombinant PCR. Luciferase assay was used to verify the direct targeted regulation of miR-127 on Bcl-6. In HepG2 cell models with overexpression or knockdown of miR-12, the changes of cell cycle and cell growth were investigated after transfection with the constructed vectors. RESULTS: The recombinant plasmids were successfully obtained as confirmed by double digestion and sequence identification. Luciferase assay showed that in 293T and HepG2 cells, miR-127 inhibited the activation of wild-type Bcl-6 3'UTR reporter vector but not mutated Bcl-6 3'UTR vector. Overexpression of miR-127 induced cell cycle arrest at G(2)/M phase and suppressed the growth of HepG2 cells, and these effects were reversed by Bcl-6 overexpression. CONCLUSION: We successfully cloned wild-type and mutated 3'UTR reporter vectors and expression vector of bcl-6 gene and confirmed their biological functions.

Comparison of form-deprived myopia and lens-induced myopia in guinea pigs.

AIM: To study the efficacy difference between form-deprived myopia (FDM) and lens-induced myopia (LIM), the degree of myopia, axial length and pathological changes of the posterior sclera from guinea pigs were evaluated. METHODS: Four-week pigmented guinea pigs were randomly assigned into 3 groups, including normal control (n=6), FDM group with monocular cover (n=11) and LIM group with monocular -7D lens treatment (n=11). FDM group was form-deprived while LIM group was lens-induced for 14 d. Refractive error and axial length were measured prior to and post treatment, respectively. Morphological changes of sclera were examined using both light and electronic microscopes. RESULTS: After 14d treatment, refractive errors for FDM group and LIM group were -3.05±0.71D and -2.12±1.29D, respectively, which were significantly more myopic than that of normal controls and fellow control eyes (P<0.01). As for axial length, it was 7.93±0.03 mm for FDM group and 7.89±0.06 mm for LIM group, which were significantly longer than both normal and fellow controls (P<0.01). With respect to both refractory error and axial length, the differences between FDM group and LIM group were not significant (P>0.05). Under light microscope, both FDM group and LIM group showed thinned sclera, disarrangement of fibrosis and enlarged disassociation between fibers. Consistently, ultrastructural examination showed degenerated fibroblasts and thinned fibers in posterior sclera. CONCLUSION: Following two weeks of myopia induction in guinea pigs, with regard to the degree of myopia, axial length and pathological alterations, there was no significant difference between FDM and LIM models. Therefore, FDM and LIM are equally effective and useful as a model of experimental myopia and guinea pigs are ideal animals for induction of experimental myopia because their high sensitivity to both form-deprivation and lens-induction.

[Inhibitory effect of lentiviral-mediated RNA on the expression of Foxp3 protein in melanoma cells].

AIM: To explore the feasibility of RNA interference in the treatment of melanoma by inhibiting the Foxp3 gene expression in mouse B16 melanoma cells using RNA interference (RNAi) in vitro. METHODS: Small interfering RNA (siRNA) was designed according to Foxp3 gene. A short hairpin RNA (shRNA) lentivirus expression vector was constructed and transfected into mouse B16 cells, and RNA interference was induced in vitro. Western blot and real-time RT-PCR were performed to detect the expression of Foxp3 gene. ELISA was applied to detect the changes of TGF-ß(1);, TGF-ß(2);, IL-10 and other cytokines. The B16 cells after interference were co-cultured with CD4(+);CD25(-);T lymphocytes. CCK8 assay was used to monitor the proliferation of CD4(+);CD25(-);T lymphocytes. RESULTS: shRNA could suppress the expression level of Foxp3, down-regulate the inhibitory ability of tumor cells on the proliferation of CD4(+);CD25(-);T lymphocytes, and reduce the secretion of TGF-ß(1);, TGF-ß(2);, IL-10 and other cytokines, in particular the expression of TGF-ß(2);. CONCLUSION: RNA interference can inhibit the expression of target gene Foxp3 in mice melanoma cells and the proliferation of tumor cells. It can also reduce the inhibition on the proliferation of CD4(+);CD25(-);T lymphocytes, and the secretion of inhibitory cytokines.

[Prokaryotic expression and purification of human GST-Cdc25C fusion protein and preliminary detection of its function].

AIM: To clone prokaryotic expression vector of Cdc25C, purify the fusion protein of GST-Cdc25C, and identify its function preliminarily. METHODS: Human Cdc25C coding region was amplified from human mammary cDNA library by PCR, and cloned into the prokaryotic expression vector pGEX-KG. The fusion protein GST-Cdc25C was expressed in E.coli Rossate and purified by GST-Sepharose 4B beads. The function of purified GST-Cdc25C was identified by GST pull-down assay. RESULTS: The GST-Cdc25C recombinant plasmid was successfully obtained by double digestion identification. The inserted fragment was confirmed correctly by sequencing. SDS-PAGE and Western blot analysis showed that the fusion protein was expressed. The fusion protein of about M(r); 80 000 was successfully induced, and identified by SDS-PAGE and Western blot analysis. GST pull-down assay showed that GST-Cdc25C could interact with Chk2 which verified its known function. CONCLUSION: Cdc25C was successfully cloned and purified.

[Prokaryotic expression and purification of human histone-1 and its activity detection].

AIM: To construct the prokaryotic expression vector of human histone-1, obtain the purified GST-H1 protein, and detect its activity. METHODS: Human histone-1 coding region was amplified from human mammary cDNA library, and was inserted into prokaryotic expression vector pGEX-KG. The recombinant plasmid pGEX-KG-H1 was transformed into E.coli Rossate. The expressed product was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis. RESULTS: The DNA fragment of about 650 bp was successfully amplified by PCR, cloned into pGEX-KG, and identified by sequencing. The recombinant protein of about M(r); 52 000 was successfully induced, purified and tested well by Kinase assay. CONCLUSION: The recombinant protein of GST-H1 is obtained successfully, which lay the foundation for further research on cell cycle control.

[Viability of corneal epithelial cells and cleavage of corneal basement after ethanol effect].

OBJECTIVE: To evaluate the viability of corneal epithelial cells and to determine the anatomic cleavage on the epithelial basement membrane after various exposure times to 20% ethanol during epithelial flap preparation in laser-assisted subepithelial keratectomy (LASEK) in cadaver eyes. METHODS: Six human cadaver eyes were exposed to 20% ethanol for 20, 30 and 40 seconds (2 eyes for each group), and another one eye was used as the control. PCNA staining was performed to determine the viability of corneal epithelial cells. Immunofluorescence staining using monoclonal antibodies against collagen VII, and immunohistological staining using monoclonal antibodies against laminin were performed to detect the anatomic location of the cleavage plane on the corneal epithelial flaps created by 20 seconds exposure to 20% ethanol in cadaver eyes. RESULTS: Hematoxylin and eosin staining of epithelial flaps revealed a coherent stratified epithelium. The PCNA positive rates of the epithelial cells in the flap decreased in the 20-second group, 30-second group and 40-second group successively. Immunohistological staining to laminin was patchy in the lifted flap and the remaining corneal basement membrane. Immunofluorescence to collagen VII, the main component of anchoring fibrils remained exclusively in the corneal bed. CONCLUSIONS: Viability of the epithelial flap decreased with longer time exposure to ethanol. The cleavage plane of the ethanol-treated corneal epithelial flap is located between the lamina lucida and the lamina densa of the basement membrane where laminin forms hemidesmosome.