| ObjectivesAIDS ( acquired immunodeficiency syndrome, AIDS ) is a immune defect disease caused by HIV (human immunodeficiency virus, HIV). Since the virus appeared in 1981 , scholar research on its mechanism . When the gp120 of virus combine with CD4 . the construct of gp120 changes to cause its hydrophobic region ( namely V3) exposed and V1 or V2 hidden inside deeply . V3 combine with the second receptor ( coreceptor ) to make gp41 merge the target cell. The scholar finds, only on terms that CD4 molecule and the second receptor exist at the same time , HIV could combine with the target cell. So besides CD4, the second receptor plays a key role in HIV infection. Studies have shown that except for CCR5 and CXCR4 , virus can also utilize some other receptors including CCR3 , CCR8 , CX3CR1 , STRL33 and orphan's receptor . CX3CR1 is a newly discovered receptor in the past two years. It's ligand, fraetalkine, can block gp120 to invade. Foreign researches recently indicate the polymorphism of CX3CR1 gene can be a marker of AIDS developing. CX3CR1 + CD8 + , CX3CR1 + CD16 + cell contain a large amount of perform and granzyme B , so the scholar presume these two cells can kill the infected cell. There are differences between Chinese's hereditary background, immune characteristic and A-merican - European crowds' , so in the world we are the first to compare the expression of CX3CR1 on CTL of the exposed seronegative individuals,the HIV infected , AIDS and the healthy, and discuss the relationship between the CX3CR1 and HIV infection,as well the progression of AIDS, now report as fol-lows.Methods1. ObjectsThe AIDS research lab of Chinese Medical University, CDC of Liaoning,Ji-lin and Henan Province confirmed 34 positive HIV/AIDS patients by Western -blot (Genelab Company) , including 15 man and 19 women , mean age is 42 years old (31 -54 years old). All patients didn't accept antivirus treatment before. 18 health control (sex , age matched with HIV/AIDS patients). 19 HIV exposed seronegative women ( measure negatively HIV antibody test , Western blot ,P24 antigen at regular intervals, and undetected virus load. There are vagina unprotect sexual intercourse average 25 times per year up to now).2. Equipment and reagentsMost of the equipment and reagents were obtained from BECTON DICKINSON company, including flowcytometer ( FACSCalibur) ; 10ml EDTA venipunc-ture vials; FACS lysing solution and the following mAbs; TriTEST CD4/CD8/ CD3 reagent, TriTEST CD3/CD16 + CD56/CD45 reagent, anti - CD8, anti -CD16, anti - 56 ( PE conjugated) and anti - CD3 ( PerCP conjugated) anti -CX3CR1 (FITC conjugated) was obtained from MBL.3. CD4 absolute value and NK cell absolute valuePipette 20 μl of TriTEST CD4FITC/CD8PE/CD3PerCP or CD3FITC/ CD56CD16PE/CD45PerCP reagent and add 50μl anticoagulated whole blood into the bottom of the TruCOUNT Tube using reverse pipetting and incubate for 15 minutes in dark at room temperature (20 -25℃ ). Add 450 μl 1X FACS Lysing Solution to the tube, incubate for 15minutes in the dark at room temperature and analyze the samples on the flow cytometer using FACS MULTISET software to acquire T lymphocyte counts,NK cell counts and corresponding ratios.4. Whole blood surface staining and FACS analysisFor each HIV infected patient, we used the following Ab combinations; CX3CR1/CD8/CD3; CX3CR1/CD16/CD3; CX3CR1/CD56/CD3; IgG2b, K/ CD8/CD3. 100 ul whole blood was added and the 6ul of each Abs were added.After a 30 - min incubation at room temperature, the erythrocytes were lysed u-sing 3ml RBC lysing solution. The cells were washed twice with PBS and resus-pended in 0.5ml PBS solution consist of 1% polyformaldehyde. There color flow - cytometric analysis was performed and FACS CELLQUEST software was used to make analysis. For subset analysis, lymphocyte were gated by forward scatter and side scatter, then CD3~+ T cells were gated and the CX3CR1 expression of CD8~+,CD16~+,CD56~+ T cells.5. Statistical analysisSPSS 11.0 software was used to make the Statistical analysis. Geometric means were determined for log - distributed variables. Comparisons of means of different groups were done using one — way ANOVA. Spearman rank correlations were used as a measure of correlation between CD4 and other variables.Results1. The expression of CX3CR1 on CD8 +T lymphocyteThe percentage of CX3CR1 + CD8 + of the infected is 18. 35 ± 10. 19% (7. 16% -36.29% ) , absolute value is 193.24 ±38.79 cell/ul(25. 88 -485. 87 cell/ul) ; the percentage of group AIDS is 23.45 ± 13. 12% (11. 73% -57. 61% ) , absolute value is 141.2 ±31.46 cell/ul(24. 87 -485.65 cell/ul) ;the percentage of ESN is 11. 05 ±6. 52% (0. 78% - 35. 61% ) , absolute value is 81.16 ± 13. 67 cell/ul(5. 35 - 194. 3 cell/ul) ; the percentage of the healthy is 5.69 ± 3. 94% (0. 33% - 14. 26% ) , absolute value is 37. 36 ± 8. 28 cell/ul (1.01 - 121. 21 cell/ul). The percentage and absolute values of the infected , AIDS patients, ESN are higher than the healthy significantly.2. The expression of CX3CR1 on CD16 +T lymphocyteThe percentage of CX3CR1 + CD16 + of the infected is 54. 11 ± 16. 38% (20.52% - 83. 39% ) , absolute value is 142. 8 ± 152. 55 cell/ul (26. 85 -607.43 cell/ul) ; the percentage of group AIDS is 54. 99 ± 13. 54% (31. 63% -76. 22% ) , absolute value is 79. 11 ±73. 15 cell/ul (18. 47 ~ 301. 33 cell/ ul) ; the percentage of ESN is 59.39 ± 16.37% (22.34% ~ 84.08% ) , absolute value is 312.49 ± 159.45 cell/ul(44. 23 -598. 11 cell/ul) ; the percentage of... |
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