The major bottle-neck in the way of constructing high density protein microarray is the availability and stability of proteins. The traditional methods of generating protein arrays require the in-vivo expression, purification and immobilization of hundreds or thousands of proteins. The cell-free protein array technology employs cell-free expression systems to produce proteins directly onto surface from co-distributed or pre-arrayed DNA or RNA, thus avoiding the laborious and often costly processes of protein preparation in the traditional approach. Here we provide an overview of recently developed novel technology in cell free based protein microarray and their applications in protein interaction analysis, in antibody specificity and vaccine screening, and in biomarker assay.
Cell-Free System ; DNA ; genetics ; Humans ; Protein Array Analysis ; methods ; Protein Biosynthesis ; Protein Interaction Mapping ; Proteins ; analysis ; genetics ; metabolism

AIM:To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transfor-mation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E.METHODS:NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at con-centrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10μmol/L).After cultured for 24 h, the mRNA expression of Ptch1, Smo,α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR.The protein levels of Shh and TGF-β1 were detected by ELISA .Immunofluorescence staining was used to evaluate the expression of Ptch 1, Smo,α-SMA, E-cadherin and type III collagen in the NRK-52E cells.RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling , and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells.Treatment with cyclopamine inhibited HH signaling by decrea-sing Smo expression and increasing Ptch 1 expression.Moreover, cyclopamine also down-regulated the expression of TGF-β1,α-SMA, type I collagen and III collagen , and up-regulated the expression of BMP-7, ZO-1 and E-cadherin.CON-CLUSION:AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells , which can be inhibited by cyclopamine treatment .The possible mechanism is that cyclopamine suppresses the activation of HH signaling , resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition .

Objective To investigate the expression of monocyte-macrophage-related factors and interstitial fibrosis in kidney tissues of rats with ureter obstruction and recanalization .Methods Forty-eight male Spragur-Dawley rats were divided randomly into the obstructive group:sham (n=6), unilateral ureteral obstruction(UUO)3 days (n=6), UUO 7 days (n=6), and UUO 14 days (n=6) and recanalization group:bilateral ureteral obstruction(RBUO)0 day (n=6), 3 days after RBUO (n=6), 7 days after RBUO (n=6), and 14 days after RBUO (n=6).The kidneys were excised on day 3, 7, and 14, and the deposition of collagen fibers in kidney was detected with HE and Masson staining . Immunohistochemical analysis was performed to evaluate the protein expressions of monocyte chemoattractant protein -1 (MCP-1), macrophage colony-stimulating factor (M-CSF) and activated-macrophage marker CD68.Real-time PCR was used to detect the mRNA expressions of MCP-1 and M-CSF.TGF-β1 levels were determined by ELISA .Results Fibrosis observed with HE and Masson staining was obviously increased in kidney tissue of UUO rats , and aggravated as time prolonged, but alleviated in rats with recanalization .TGF-β1 levels were increased obviously in the UUO group , but decreased in rats with recanalization compared with those in BUO rats .In UUO rats, mRNA and protein expression levels of MCP-1 and M-CSF were increased .MCP-1 and M-CSF expression was gradually decreased in rats with recanalization compared with those in BUO rats .The dynamic change in expression of MCP-1 and M-CSF in both UUO rats and recanalization rats was consistent with the change in expression of CD 68. Conclusion Dynamic change in expression of MCP-1 and M-CSF in kidney tissues reflects change of activated and accumulated monocyte -macrophages , which may be one of the major mechanisms contributing to fibrosis induced by ureter obstruction .Renal fibrosis is alleviated by down-regulated expression of monocyte-macrophages factors with recanalization operation .

The gammac cytokines play an important role in proliferation and survival of T cells. Blocking the gammac signals can cause the activated donor-reactive T cells losing the ability to proliferate, and getting into apoptosis pathway, which contributes to induction of the peripheral tolerance. In this study, we induced the transplant tolerance through blocking the gammac in combination with donor-specific transfusion (DST) in the cardiac transplantation. Following DST, on the day 2, 4 and 6, C57BL/6 recipients received anti-gammac monoclonal antibodies (mAbs) injection, and those in control group were not given anti-gammac mAbs. On the day 7, Balb/c cardiac allografts were transplanted. All recipients in experimental group accepted cardiac allografts over 30 days, and two of them accepted allografts without rejection until sacrifice on the 120 day. Animals only receiving DST rejected grafts within 5 days, and the mice receiving cardiac transplantation alone rejected grafts within 9 days. Our study showed that blockade of gammac signaling combined with DST significantly prolonged allograft survival, which was probably associated with inhibition of antigen-specific T-cell proliferation and induction of apoptosis.

Objective:To study the feasibility of transplantation of normal rat penile corpus cavernosum and major pelvic ganglion (MPG)into the renal subserous region of a Nu /Nu mouse based on allograft technology.Methods:Penile corpus cavernosum and MPG,harvested from Sprague-Dawley (SD)rats under sterile condition,were transplanted underneath the kidney capsule of Nu /Nu mice through the mi-crosurgery instruments and surgery microscope.The histopathologic changes and cellular proliferation in the transplanted penile corpus cavernosum and MPG were then analyzed at the end of 1week and 4 weeks after transplantation.Histological staining and immunohistochemical staining were used to evaluate the main outcome measures.Results:After 1 week,the tissue morphology of the transplanted corpus caverno-sum underneath the kidney capsule of Nu /Nu mice was consistent with normal penile corpus cavernosum, and blood could be observed in the penis cavernous sinus of the graft;after 4 weeks,the mophorlogy of the tranplanted corpus cavernosum near the kidney was consistent with normal penile corpus cavernosum, while fibrosis was noteworthy in the graft away from the kidney,but blood could still be seen in the penis cavernous sinus.After 1 week,the tissue morphology of the transplanted MPG was consistent with normal MPG,multiple islet-like cell clusters could be seen in the transplanted MPG in the renal subserous re-gion,and angiogenesis could be observed near the kidney;after 4 weeks,a network of blood vessels was clearly visible away from the kidney,and islet-like cell clusters were still clearly observed in the trans-planted MPG.In addition,ki67 positive cells were observed in the transplanted penile corpus cavernosum and MPG after 4 weeks of transplantation,which indicated that there was still cell proliferation activity in the grafts.Conclusion:The transplanted corpus cavernosum and MPG underneath the kidney capsule of Nu /Nu mice could survive at least 4 weeks.Moreover,the inner structure of the transplanted corpus ca-vernosum and MPG was close to the normal tissue.The underlining mechanism may be related to the lo-cal microenvironment underneath the kidney capsule of Nu /Nu mice and the neovascularization in the transplanted grafts.

The γc cytokines play an important role in proliferation and survival of T cells. Blocking the γc signals can cause the activated donor-reactive T cells losing the ability to proliferate, and getting into apoptosis pathway, which contributes to induction of the peripheral tolerance. In this study, we induced the transplant tolerance through blocking the γc in combination with donor-specific trans-fusion (DST) in the cardiac transplantation. Following DST, on the day 2, 4 and 6, C57BL/6 recipients received anti-γc monoclonal antibodies (mAbs) injection, and those in control group were not given anti-γc mAbs. On the day 7, Balb/c cardiac allografts were transplanted. All recipients in experimental group accepted cardiac allografis over 30 days, and two of them accepted allografis without rejection until sacrifice on the 120 day. Animals only receiving DST rejected gratis within 5 days, and the mice receiving cardiac transplantation alone rejected gratis within 9 days. Our study showed that blockade of γc signaling combined with DST significantly prolonged allografi survival, which was probably associated with inhibition of antigen-specific T-cell proliferation and induction of apoptosis.