Objective To observe the effect of the advanced glycation end products(AGEs)on the iNOS expression in cultured cardiac microvascular endothelial cells.Methods Cultured the cardiac microvascular endothelial cells in vitro.After AGEs of the different dose(50～200 mg/L)and different times(0～24 h)played the role on the cells,we determined the NO generation and iNOS protein expression of all groups.Results NO generation and iNOS protein expression increased with the AGEs-dose increasing and treatment time.The results were significantly different.Conclusion These results demonstrate that AGEs may induce iNOS to produce toxic NO in cardiac microvascular endothelial cells,then the diabetic cardiomyopathy occures.

OBJECTIVE:To study the effect and its possible mechanism of Xuezhiping capsule on blood lipid levels of high blood lipid model golden hamsters. METHODS:Golden hamsters were randomly divided into normal control group,model group, atorvastatin group (3 mg/kg),combination group (Xuezhiping capsule 1.3 g/kg+atorvastatin 1.5 mg/kg),Xuezhiping capsule low-dose,medium-dose,high-dose groups(Xuezhiping capsule 1.3,2.6,5.2 g/kg),10 in each group. Hamsters in normal control group received normal diet,the other groups were given high-fat diet to establish high blood lipid model. Then relevant drugs were intragastrically given 2 weeks later. Normal control group and model group were intragastrically given equal volume of distilled wa-ter,once a day,for 4 weeks. After last administration,blood lipid indexes(TG,TC,HDL-C,LDL-C,FFA),mRNA and protein expressions of lipid metabolism related genes (PPAR-α,CYP7A1,ACOX,SREBP-1c,ACC1) were detected. RESULTS:Com-pared with normal control group,serum contents of TC,TG,LDL-C,FFA in model group increased,HDL-C content decreased;PPAR-α,CYP7A1,ACOX mRNA and protein expressions were weakened,SREBP-1c,ACC1 mRNA and protein expressions were enhanced(P<0.05). Compared with model group,above-mentioned indexes were obviously improved in Xuezhiping capsule high-dose group,atorvastatin group and combination group (except for HDL-C in Xuezhiping capsule high-dose group)(P<0.05);TC,TG,FFA contents,and PPAR-α,CYP7A1,ACOX,ACC1 mRNA,and CYP7A1,SREBP-1c,ACC1 protein were obviously improved in Xuezhiping capsule medium-dose group(P<0.05);TC,TG contents,and PPAR-α mRNA,and CYP7A1, SREBP-1c protein were obviously improved in Xuezhiping capsule low-dose group (P<0.05). CONCLUSIONS:Xuezhiping cap-sule has a promising effect of lowering blood lipid,the mechanism may be related to activating PPAR-α and inhibiting SREBP-lc signaling pathway.

Objective To analyze the virologic and immunologic properties during SHIV-XJ02170passage in vivo and construct the clade C SHIV/Chinese origin Rhesus macaques AIDS model . Methods SHIV-XJ02170 cell-free virus tranfected in 293T was adapted by serial passage in nine Chinese-origin Rhesus macaques. CD4/CD8 ratio was detected by flow cytometry to analyze the changes in viral pathogenicity. Real-time RT-PCR and IFN-γsecreting ELISPOT methods were used to analyze changes in characteristics of virology and immunology. Results During in vivo passage, CD4/CD8 ratio did not deeply decline. However,the peak and setpoint viral load in the line 3 show a continuous upward trend. The strong humoral and cellular immune responses were induced after SHIV-XJ02170 infection. Meanwhile, there was significant positive correlation between the viral load and binding antibody titer. Conclusion There were no pathogenic viral strains, and upward trend in virulence of SHIV-XJ02170 was found during in vivo passaging. SHIVXJ02170/Chinese origin Rhesus macaques model will play an important role in effect evaluation of candidate AIDS vaccines in China.

SHIV-CN97001 played an important role in assessing the immune effect and strategy of the AIDS vaccine which included genes of the predominant prevalent HIV-1 strain in China. In this study, SHIV-CN97001 was in vivo passaged serially to construct pathogenic SHIV-CN97001/rhesus macaques model. To identify variation in the gp120 region of SHIV-CN97001 during passage, the fragments of gp120 gene were amplified by RT-PCR from the plasma of SHIV-CN97001 infected animals at the peak viral load time point and the gene distances (divergence, diversity) were calculated using DISTANCE. The analysis revealed that the genetic distances of SHIV-CN97001 in the third passage animals were the highest during in vivo passage. It had a relationship between viral divergence from the founder strain and viral replication ability. The nucleic acid sequence of the V3 region was highly conservative. All of the SHIV-CN97001 strains had V3 loop central motif (GPGQ) and were predicted to be using CCR5 co-receptor on the basis of the critical amino acids within V3 loop. These results show that there was no significant increase in the genetic distance during serial passage, and SHIV-CN97001 gp120 gene evolved toward ancestral states upon transmission to a new host. This could partly explain why there was no pathogenic viral strain obtained during in vivo passage.

Objective To identify the variation in the Env region of SHIV-XJ02170 during passaging in Chinese origin Rhesus Macaques.Methods Fragments of the SHIV-XJ02170 gp160 and gp120 gene were amplified by PCR and RT-PCR separately from the blood samples of SHIV-XJ02170 infected animals at the peak viral load time point.Purified RT-PCR product was ligated into T easy vector and transformed into JM109 competent cells,18 clones were selected by PCR method and sequenced by ABI 3730DNA sequencers.The gene distances(divergence,diversity)were calculated using DISTANCE.Results In all,the SHTV-XJ02170 gp120 gene evolved forward along the virus passaging.It could be found that viral divergence from the founder strain serially enhanced during in vivo passaging,but in the early phase of each passage,SHIV-XJ02170 gp120 gene evolved toward ancestral state upon transmission to a new host.All of the SHIV-XJ02170 strains had V3 loop central motif(GPGQ)and were predicted to be using CCR5 on the basis of the critical amino acids within V3 loop.Conclusion There was significant increase in the genetic distance during serial passaging,and SHIV-XJ02170 gp120 gene evolved forward along passaging.This could partly explain why the virus infectivity was enhanced during in vivo passaging.

Objective To compare the infectivity between laboratory adapted human inununodefi- ciency virus(HIV-1) and primary HIV-1 isolates for different mucosal epithelial cell lines. Methods Mu-cosal epithelial cells Caco-2, T-84, HeLa and lymphocyte MT-4 were infected with laboratory adapted HIV-1 SF33 and 2 primary HIV-1 isolates (02010561, 02010141). Culture supernatant and cells were collected respectively on 3-4 days interval after virus inoculation. The former was tested for HIV-1 antigen P24 level and viral load, and the latter was tested for total viral DNA and integrated viral DNA. Results All 3 virus strains could infect MT-4 cells and integrate into their genome. Only HIV-1 SF33 could infect Caco-2 cells but could not integrate into their genomic DNA. Both HIV-1 SF33 and 02010561 infected HeLa cells but only integration of HIV-1 SF33 was detected. All the 3 HIV-1 strains infected T-84 cells but only the integra-tion of HIV-1 SF33 and 02010141 was observed. Conclusion Although laboratory adapted and primary HIV-1 strains are able to infect human mucosal epithelial cell lines, transient or productive infection estab-lished in different mucosal epithelial cells is dependent on the character of cells and virus strains.

Objective To compare the mRNA level of macrophage inflammatory protein-3α(MIP-3α) in 3 different mucosal epithelial cell lines. Methods RNA standards were prepared by in vitro transcription. One-step real-time RT-PCR was established and optimized using TaqMan EZ RT-PCR Core Reagents with TaqMan probes and primers specific to human MIP-3α mRNA sequence. The specificity of one-step real-time RT-PCR method was confirmed by sequencing the PCR products. The sensitivity and reproducibility of the method was examined by repeating the test 8 times with the same sample. Results The one-step real-time RT-PCR with a wide detection range is sensitive, reproducible. It was found that MIP-3α mRNA level in Caco-2 and T-84 cells was much higher than that in the HeLa cells. Conclusion High level of MIP-3α mRNA could be found in mucosal epithelial cells and difference in transcription level of MIP-3α may exist in epithelial cells from different mucosa.

Objective To compare the levels of indoleamine 2,3-dioxygenase (IDO) in the peripheral blood mononuclear cells(PBMCs) from HIV-1 infected and HIV-1 negative individuals and in human tumor cells in the presence or absence of TLR ligand stimulation.Methods TaqMan probe real-time RT-PCR method for human IDO mRNA was established; IDO mRNA levels in the PBMCs from HIV-1+ and HIV-1-individuals were tested; IDO mnRNA levels in mucosal origin(T84,Caco-2,Hela) and leukocyte origin(THP-1,MT-4) tumor cells before and after exposure to agonists for TLR4,TLR7/8 and TLR9 were examined.Results It was found that a high level of IDO mRNA could be found in HIV-1+ individuals( 103.42 copy IDO mRNA/106 copy GAPDH mRNA) ; however,some high risk HIV-1-individuals may have also a high level of IDO mRNA.Some of the tumor cells could express higher level of IDO mRNA after exposure to TLR agonist.Conclusion This study indicated a role for IDO in the viral persistence and tumor formation in HIV/AIDS and further studies were warranted.

OBJECTIVETo study the expression of Toll-like receptors (TLRs) mRNA in human trophoblast HTR-8/SVneo cells and the changes in indoleamine 2,3-dioxygenase (IDO) mRNA expression in response to TLR ligand stimulation.

METHODSThe expressions of TLRs and IDO mRNA in human HTR-8/SVneo cells were tested by RT-PCR, and the changes in IDO mRNA levels after exposure to TLR3, TLR4, TLR7/8, and TLR9 ligands were quantitatively analyzed with real-time PCR.

RESULTSIDO and TLR1-10 mRNAs were expressed in HTR-8/SVneo cells. As the cell culture time extended, IDO mRNA expression level tended to increase within 48 h. After stimulation with the TLR ligands, the expression of TLR-3 mRNA was down-regulated while the expression of TLR-4, 7, 8, and 9 mRNA up-regulated. Stimulation of the cells with poly(I:C) lowered the expression of IDO mRNA while IFN-γ increased its expression.

CONCLUSIONSThe expression of IDO mRNA is associated with the nutrition of the maternal-fetal interface. Stimulation with the TLR ligands affects the expression of IDO and TLR mRNA expressions in the cells, which verifies the functional activity of TLRs and suggests a role of IDO in TLR pathway-dependent antiviral immunity.

Cell Line ; Female ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; genetics ; metabolism ; Interferon-gamma ; pharmacology ; Ligands ; Poly I-C ; pharmacology ; RNA, Messenger ; metabolism ; Toll-Like Receptors ; genetics ; metabolism ; Trophoblasts ; cytology ; metabolism