Establishment and Comparison of Two Mouse Models of Celiac and Cervical Heterotopic Heart Transplantation / 中国实验动物学报

Objective Objective In order to better keep up with the development of transplantation immunobiology.we established and compared two types of mouse heterotopic heart transplantation,and hope to help further organ transplantation studies.Methods According to the surgical procedures of Ono's type and Chen's type of mouse model of heterotopic heart transplantation with some modification,we performed celiac and cervical heteropotic heart transplantation between iso-strains and hetero-strains,and compared the operation suecess rate,operation time,allografi survival time,and histopathology of those establishment methods.Results The success rates of mouse celiac and cervical heterotopic heart transplantation were 86.7% and 83.3%,respectively,with a non-significant difference(P>0.05) between the two methods of operation regarding the total operation time,survival time of the allografts,and histopathological findings.Conclusions Based on the mastery of microsurgical techniques,the two models of heterotopic mouse heart transplantation can be established equally,and either of them can be considered depending on the particular requirements of studies.

Establishment of stable high expression cell line with green fluorescent protein and resistance genes / 华中科技大学学报（医学）（英德文版）

In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

Establishment of Stable High Expression Cell Line with Green Fluorescent Protein and Resistance Genes / 华中科技大学学报（医学）（英德文版）

In order to establish stable high expression cell lines, the eukaryotic expression vector pIRES2EGFP and recombinant plasmid pIRES2EGFP-TIM-3 were transfected into mammalian cells CHO by Lipofectamine. The transfected cells were cultivated under selective growth medium including G418 and green fluorescent protein (GFP) positive cells were sorted by FACS. Simultaneously, growing transfectants were selected only by G418 in the medium. The GFP expression in stably transfected cells was detected by FACS. Under selective growth conditions with G418, the percentage of GFP positive cells was reduced rapidly and GFP induction was low. In contrast, the percentages of GFP positive cells were increased gradually after FACS. By 3 rounds of GFP selection, the stable high expression cell lines were established. Furthermore, using FACS analysis GFP and the target protein TIM-3 co-expression in the stable transfectants cultured in nonselective medium was detected. Theses results demonstrated that the stably transfected cell lines that express high titer of recombinant protein can be simply and fleetly obtained by using GFP and selective growth medium.

Studies on activity of NK cells in preeclampsia patients / 华中科技大学学报（医学）（英德文版）

The activity of the NK cells in patients with preeclampsia was studied to investigate the pathogenesis of preeclampsia. By using MTT and 51Cr releasing technique, the proliferation and killing ability of the NK cells in maternal and umbilical blood from preeclampsia patients (n = 18) and normal third trimester pregnant women (n = 18) were detected. The NK-92 cell line was as the positive control. The results showed that the NK cell counts of umbilical blood in preeclampsia patients and normal third trimester pregnant women were significantly greater than those of maternal blood (both P<0.05). Compared with that in normal third trimester pregnant women, the proliferative ability of the NK cells in preeclampsia patients was apparently increased (P<0.05). Compared with that in maternal blood, the proliferative ability of the NK cells in umbilical blood from both preeclampsia patients and normal third trimester pregnant women was dramatically increased. The killing ability of the NK cells in preeclampsia patients was significantly higher than that in normal third trimester pregnant women (P <0.05). It was suggested that both number and function of the NK cells in preeclampsia women were increased, and that in umbilical blood was greater than that in maternal blood, speculating that the function of the NK cells may affect the maintenance of the maternal and fetal immune tolerance during pregnancy.

Studies on activity of NK cells in preeclampsia patients / 华中科技大学学报（医学）（英德文版）

The activity of the NK cells in patients with preeclampsia was studied to investigate the pathogenesis of preeclampsia. By using MTT and 51Cr releasing technique, the proliferation and killing ability of the NK cells in maternal and umbilical blood from preeclampsia patients (n = 18) and normal third trimester pregnant women (n = 18) were detected. The NK-92 cell line was as the positive control. The results showed that the NK cell counts of umbilical blood in preeclampsia patients and normal third trimester pregnant women were significantly greater than those of maternal blood (both P<0.05). Compared with that in normal third trimester pregnant women, the proliferative ability of the NK cells in preeclampsia patients was apparently increased (P<0.05). Compared with that in maternal blood, the proliferative ability of the NK cells in umbilical blood from both preeclampsia patients and normal third trimester pregnant women was dramatically increased. The killing ability of the NK cells in preeclampsia patients was significantly higher than that in normal third trimester pregnant women (P <0.05). It was suggested that both number and function of the NK cells in preeclampsia women were increased, and that in umbilical blood was greater than that in maternal blood, speculating that the function of the NK cells may affect the maintenance of the maternal and fetal immune tolerance during pregnancy.

Construction of antisense RNA expression plasmid for u-PAR and its transfection to highly invasive PC-3M cell subclones / 华中科技大学学报（医学）（英德文版）

To evaluate the specific inhibition of antisense u-PAR on the u-PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u-PAR obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u-PAR recombinant was transfected into highly invasive cell subclones. The u-PAR expression in neo-resistant cells was examined by RT-PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u-PAR in transfected cells decreased sharply, and the rate of inhibition was 53% and 73%, respectively, indicating that an antisense u-PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u-PAR on invasion in highly invasive cell subclones of human prostate carcinoma.

The inhibitory effects of an antisense u-PAR vector on invasion of highly invasive human prostate carcinoma PC-3M cell subclones / 华中科技大学学报（医学）（英德文版）

To observe the inhibitory effects of an antisense u-PAR vector on invasion of highly invasive PC-3M cell subclones, the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones were detected by a quantitative RT-PCR and zymography. The monolayer invasion assay and colony formation assay in soft agar were used. And tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. It was found that in vitro growth of highly invasive PC-3M cell subclones transfected with the antisense u-PAR was declined, and the ability of anchorage-independent growth of those cell subclones was found decreased sharply, with the inhibiting rate becoming 79% and 60%, respectively. Although the antisense u-PAR didn't change MMP-9 gene transcription, they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups was statistically significant (P < 0.01). The antisense u-PAR vector could not only inhibit the invasion ability of highly invasive PC-3M cell subclones in vitro but also restrain the growth of those cell subclones in vivo.

Antitumor effects of the fibroblasts transfected TNF-alpha gene and its mutants / 华中科技大学学报（医学）（英德文版）

To compare the anti-tumor effects of transmembrane TNF-alpha (TM-TNF) and secreted TNF-alpha (S-TNF) in vivo, mouse fibroblasts NIH3T3 were transfected separately with three types of retrovirus containing wild type TNF-alpha (Wt-TNF), TM-TNF mutant (TM-TNFm), S-TNF mutant (S-TNFm). Southern blot, RT-PCR, FACS and bioassay were used to investigate TNF-alpha gene integration, expression and its biological activity. It was found that both fixed cells and supernatant of NIH3T3/Wt-TNF, the fixed cells of NIH3T3/TM-TNFm and the supernatant of NIH3T3/S-TNFm could express high level of TNF-alpha or its mutants and effectively kill H22 in vitro. The transfected NIH3T3 were separately injected into the mice at the sites of H22 tumor cell inoculation according to a ratio of 5:1 or 1:1 (effector/target cells, E/T) after the third day of H22 challenge, respectively. At the E/T = 5:1, the NIH3T3/TM-TNFm induced the highest tumor regression, while NIH3T3/S-TNFm exerted the strongest tumor depressing effect at the E/T = 1:1 in vivo. No obvious side effects were noted throughout the course of treatment. The results suggest that both TM-TNF and S-TNF could cause tumor regression. The anti-tumor effect of TM-TNF would be more powerful and safe than that of S-TNF at the proper E/T ratio.

Study of the effects of LPS on the TACE gene expression and its function / 华中科技大学学报（医学）（英德文版）

In order to investigate the effects of LPS on the TACE gene transcription and expression and its regulating effect on the TM-TNF secretion, in vitro studies were carried out on HL-60 cells stimulated by LPS. TACE, TNF-alpha mRNA levels were detected by Dot-Elisa and the distribution of membrane molecules determined by flow cytometry assay and indirect immunofluorescence. The results showed that: (1) TACE was detected in or on HL-60 cells and it is predominantly localized on cell surface and to a perinuclear compartment. (2) LPS induced a time dependent increasement of TNF-alpha mRNA and enhanced TNF conversion with decreasing distribution of TNF in cell surface and increasing secretion of TNF protein. Such conversion could be inhibited by TACE ODN. (3) LPS also induced time-dependently increased expression of TACE gene and activation of its function. On the other hand, TACE protein in cell lysate and on cell surface was decreased. It was suggested that TACE molecular structure might change following its mediating membrane-anchored molecular secretion.

Possible association of HLA-DRB1 gene with the autoantibody against myocardial mitochondria ADP/ATP carrier in dilated cardiomyopathy / 华中科技大学学报（医学）（英德文版）

To probe the genetic background and immunopathogenesis of dilated cardiomyopathy (DCM) 77 patients with DCM, HLA-DRB1 gene polymorphism were analyzed by using the polymerase chain reaction/sequence specific primer (PCR/SSP) technique and autoantibody against myocardial mitochondria ADP/ATP carrier were examined by using the Immunoblot analysis. The frequency of HLA-DRB1*0901 allele was significantly higher in DCM patients in which autoantibody against ADP/ATP carrier of myocardial mitochondria is positive in contrast with those in which the autoantibody is negative (25.46% vs 3.45%, P < 0.05), the relative risk (RR) being 9.56. The other frequencies of HLA-DRB1 alleles have no significant difference in the antibody positive group and negative group. It is possible that a subset of DCM patients may exist in which autoimmunity is associated with genetic factors.

Molecular cloning and sequencing of the cDNA of the soluble human leukocyte antigen-G1 / 免疫学杂志

Objective　To construct a recombinant plasmid pcDNA3-sHLA-G1 expressing soluble HLA-G1. Methods　 Total cell RNA was extracted from the cell line Jeg-3 and the cDNA was amplified by RT-PCR； The cDNA fragment was inserted into the eukaryotic expressing vector pcDNA3 and the recombinant plasmid was identified by restriction endonucleases digestion and sequencing. Results　 After restriction endonucleases treatment and sequencing, it was confirmed that the pcDNA3-sHLA-G1 had been constructed successfully. Conclusion　 In this study, the recombinant plasmid pcDNA3-sHLA-G1 had been constructed successfully.

Effect of transfection of tumor necrosis factor ? gene and its mutants on tumorigenicity of H22 tumor cells in vivo / 中国病理生理杂志

AIM: To compare the tumorigenecity of H22 cells transfected with TNF-? gene and its mutants (secreted TNF-? mutant, S-TNFm, transmembrane TNF-? mutant, TM-TNFm and wild type of TNF-?, Wt-TNF) in vivo . METHODS: Three kinds of mouse liver cancer cell line H22 expressing TNF-? and its two mutants were mixed with untransfected H22 at different effector/target ratio separately. The growth of tumor was examined after injection of 2.5?10 5 (100 ?L) mixed H22 tumor cells into mice. The lymphocyte infiltration in the site of tumor and the expression of Fas on tumor cells were detected by immunohistochemistry. RESULTS: The tumorigenecity of H22 cells transfected with TNF-? gene and its mutants was significantly weakened ( P

The effect of secreted and transmembrane stem cell factor on biological activities of mast cells / 中国免疫学杂志

Objective:To study the effects of secreted stem cell factor(S SCF) and transmembrane SCF(TM SCF) on the biological functions of mast cells,including proliferation,release of histamine and secretion of cytokine to explore the biological characteristics of the two forms of SCF.Methods:MTT,fluorescence technique,in situ hybridization were used to detect the several biological functions of mast cells,such as proliferation,release of histamine and the transcription of TNF ? mRNA.Results:It was shown that S SCF could significantly promote the proliferation of mast cells,release of histamine and TNF ? mRNA transcription,while TM SCF had only a weaker positive effect on histamine relase and no any effect on proliferation and TNF ? mRNA transcription.Conclusion:The biological actions of TM SCF on mast cells are different from that of S SCF.

The Inhibitory Effects of an Antisense u-PAR Vector on Invasion by Highly Invasive Human Prostate Carcinoma PC-3M Cell Subclones / 中国肿瘤生物治疗杂志

Objective: To observe the inhibiting effects of an antisense u-PAR vector on invasion by highly invasive PC-3M cell subclones. Methods: The effects of an antisense vector on invasion by highly invasive PC-3M cell subclones were observed and compared in vitro by monolayer invasion assay and soft agar clone. Then, both a quantitative RT-PCR and zymography were used to exam the effects of the antisense u-PAR on activity of MMP-9 in those highly invasive cell subclones. Furthermore, the tumorigenesis rate and invasions by the cell subclones with or without the antisense u-PAR were observed in nude mice. Results: It is found that the speed of growth in vitro was slowing down by highly invasive PC-3M cell subclones transfected with the antisense u-PAR, and the ability of anchorage-independent growth of those cell subclones was also decreasing sharply,and the inhibiting rate was 79% and 60%, respectively. Although the antisense u-PAR didn′t change MMP-9 gene transcription, but they could inhibit the activation of MMP-9 of highly invasive PC-3M cell subclones. Moreover, the tumorigenesis rate of the cell subclones with the antisense u-PAR decreased and the growth of a neoplasm also slowed down. The t tests showed the difference between experimental and control groups reached statistical significance ( P

Comparison of Antitumor Effect in vivo between Transmembrane TNF-? and Secretory TNF-? / 中国肿瘤生物治疗杂志

Objective: To study the antitumor effects of transmembrane TNF-? and secretory TNF-? in vivo. Methods: Three types of TNF-? cDNA plasmids (wild type TNF-?; transmembrane TNF-? mutant; secretory TNF-? mutant) were directly injected into tumor-tearing mice. Results: The three types of TNF-? could be expressed by tumor cells and all of them could inhibit evidently the rate of tumor growth. The tumor regression after treatment with transmembrane TNF-? mutant at the early stage was more significant than that with the other two types of TNF-?( P

Dependence of Transmembrane TNF-? Bioactivity on Biomembrane / 中国肿瘤生物治疗杂志

Transmembrane TNF-? is an integral protein expressed on the surface of avtivated monocytes/macrophages. In order to define the interrelation between TM-TNF and biomembrane, we used an in vitro translation system and microsomal membranes to engender expression models of two types of TM-TNF; TM-TNF anchored to microsomes and naked 26kD TNF without membranes. When the cytotoxic activity of these two types of TNF was compared, the synthesized 26kD TNF was found to exert its effects only in the presence of microsomal membranes. With the use of indirect immunofluorescence technique, we further studied the binding of the in vitro translated 26kD TNF with TNFR on the membrane of HL60 cells. It was found that only TM-TNF attached to the microsomes could bind effectively with TNFR, while naked 26kD TNF could not. These results suggest that binding with biomembrane may be the prerequisite for 26kD TNF to fold properly for displaying its biological effects. In case 26kD TNF is freed from membrane, it can no more bind with TNFR, there by leading to loss of its cytotoxic effect.

Expression of HLA class Ⅱ antigens, interleukin-2 receptors and transferrin receptors of monocytes from patients with insulindependent diabetes mellitus / 中国病理生理杂志

Expression of HLA class Ⅱ antigens(HLA-DR, DQ and DP), interleukin2 receptors(IL-2R) and transferrin receptors(TfR) of blood monocytes from 10 patients with insulin-dependent diabetes meIlitus (IDDM) were assayed with the indirect immune fluorescence technique using corresponding monoclonal antibodies and the FITC-labelled second antibody. The results showed that the number of HLA-DQ~+ monocytes was much more in diabetics than in normal controls. The percentages of HLA-DR~+ and HLA-DP~+ monocytes in diabetics were not different significantly from those in normal controls. Besides, IL-2R~+ and TfR~+ monocytes were also found to be very much increased in diabetics as compared with controls. It was possible that increased expression of HLA-DQ antigen, IL-2R and TfR of monocytes in patients with IDDM might play a role in the pathogenesis of the autoimmune reaction.

Effect of substance P on the expression of HLA-DR and HLA-DQ antigens on human monocytes / 中国病理生理杂志

Recent studies have shown that the the nervous system possesses immunoregulatory function. We investigated the effect of substance P(SP) a neuropeptide on the expression of HLA-DR and HLA-DQ antigens on normal human monocytcs of the peripheral blood. APAAP (alkaline phosphatase-antialkalinc phosphatasc) enzyme immunoassay was adopted to detect the HLA class Ⅱ molccults, using monoclonal antibodies Tu36, Tu22, in the reaction system. The results, showed that SP at concentrations higher than the physiological serum level (10~(-10)mol/L, 30~(-8)mol/L, 10~(-6)mol/L) promoted the expression of HLA-DQ antigen on monocytcs significantly in vitro (P0.05). The maximal effect was observed at 10~(-8)mol/L. Expression of HLA-DR antigen, however, was not influenced by SP in the same range of SP concentrations. It is reasoned by the authors that the immunosupprcssivc effect of excessive glucocorricoids during stress might partly be counterbalanced by the uprcgulatory action of increased blood SP on the immune function.

In vivo effect of antiinflammatio No.6, streptomycin and chloromycetin on granulocyte migration in the rat / 中国病理生理杂志

Daily i.v. injection of 4ml of the traditional Chinese medicine antiinflammatio No.6(AI6) to rats for 3 days resulted in a marked acceleration of chemotaxis (Cht) of neutrophil granulocytes(NG). Daily i.m. injection of streptomycin(SM) and chloromycetin(CM), each in a dose of 50mg per animal, given to rats for 3 days led to a marked depression of Cht of NG, while combined with daily i.v. injection of 4ml of AI6 caused not only a complete cancelling of the suppressive effect of the antibiotics on NG Cht, but also a marked acceleration of NG Cht. Combined application of AI6 and certain antibiotics in the clinical treatment of certain acute bacterial infections is suggested.

The relationships between structure and function of human transmembrane tumor necrosis factor-? / 中国免疫学杂志

Objective:To determine the region of human transmembrane tumor necrosis factor alpha (TM TNF?),essential for cytotoxic activity against mouse L929 cell.Methods:Single amino acid substituted TM TNF? mutant proteins(muteins) were produced by in vitro transcription linked translation techniques.An expression cDNA for TM TNF? was site directed mutagenized by recombinant PCR.7 single amino acid substituted TM TNF? muteins were generated and assayed for cytotoxic activity.Results:The cytotoxic activities of TM TNF? muteins,eg,TM TNF? -71/Lys was