Objective To investigate the regulatory effect of CsA on the expression of NK cell inhibitory receptor ILT4 and cytotoxicity of NK cells.Methods NK cells treated with CsA ( 10 mg/L) or DMSO for 12,24 and 36 h were chosen as three experimental groups and control groups respectively.RTqPCR and flow cytometry were performed to detect the alteration of ILT4 at the mRNA and protein level respectively.The expression of HLA-G in human gastric cancer cell line BGC-823 and human placental choriocarcinoma cell line JEG-3 were measured at the same time,and then the cytolytic activity of the untreated NK cells and NK cells treated with CsA for 36 h against BGC-823 and JEG-3 cells was determined with MTT.One-way analysis of variance was employed to compare the different ILT4 expression at different time points after medication; Dunnett test was performed to carry out the pairwise comparison between each mean.The difference of HLA-G expression between JEG-3 cells and BGC-823 cells,and the difference of NK cell cytolytic activity against JEG-3 cells and BGC-823 cells were analyzed by student's t-test.Results RT-qPCR assay indicated that the relative levels of ILT4 mRNA in NK cells treated with CsA for 12,24 and 36 h in turn were 0.99 ± 0.27,1.79 ± 0.29,6.79 ± 0.64,and those of their contrast groups treated with DMSO were 0.86 ±0.11,0.94 ±0.12,1.06 ±0.17.The expression of ILT4 in NK cells treated with CsA for 24 h or 36 h was higher than that in NK cells of their contrast groups respectively ( t value of 4.69,14.99,P ＜0.05,respectively),but there was no significant difference between the two groups of NK cells treated for 12 h ( t =0.78,P ＞0.05 ).Through flow cytometry,the positive rates of ILT4 protein expression in NK cells treated with CsA for 12,24 and 36 h [(5.16 ± 0.42 ) ％,( 6.23 ± 0.48 ) ％,( 23.8 ± 1.5 ) ％]were higher than those in NK cells after treatment with DMSO for 12,24 and 36 h respectively[(3.08 ±0.19)％,(3.35 ±0.12)％,(3.36 ±0.21 )％ ;t value of 7.70,10.06,20.72,P＜0.01,respectively].The expression of ILT4 in NK cells treated for 36 h was much higher than that in NK cells for 12 and 24 h at the mRNA and protein level (t value of 16.38,14.12 ;21.81,20.56,P ＜ 0.01,respectively).Meanwhile the killing rates of NK cells treated with 10∶1 effector-target ratio CsA on BGC-823 cells (low HLA-G expression) were ( 8 1.96 ± 2.80 ) ％ ( before treatment) and ( 60.23 ± 1.57 ) ％ ( after treatment),which were higher than those on JEG-3 cells (HLA-G-overexpression) [(53.46 ±2.21 )％ ( before treatment),(28.30 ± 1.85 ) ％ ( after treatment)].The changes of cytotoxicity of NK cells treated with CsA against target cells showed that CsA inhibited the killing activity of NK cells to BGC-823 and JEG-3 cells (t value of 11.74,15.16,P＜0.01,respectively),and the inhibitory rates were (26.48 ±2.42)％ and (47.10 ±1.59 ) ％ respectively.CsA had a higher killing rate inhibition on JEG-3 than on BGC-823 ( t =12.31,P ＜0.01 ).Conclusion CsA induces upregulation of ILT4 in NK cells,and the cytotoxicity of NK cells to tumor cells can be affected by interaction of ILT4 and HLA-G.

Objective To study the correlations between genetic polymorphisms of TNF-α as well as IL-6 and susceptibility to colorectal cancer among Chinese Han people in Shandong province.Methods Single nucleotide polymorphisms (SNPs) of TNF-α-238G/A,-308G/A and IL-6-174G/C,-572G/C,-597G/A in 490 patients with colorectal cancer were analyzed by using gene chip.Concentrations of TNF-α and IL-6 in serum samples were measured by ELISA.A case-control study was conducted to analyze the correlations between SNPs of TNF-α-238G/A,-308G/A as well as IL-6-174G/C,-572G/C,-597G/A and susceptibility to colorectal cancer.Chi-square test or t test was used for statistical analysis.Relative risks were estimated based on the values of odds ratio (OR) and 95% confidence interval (95%CI).Results The frequency of TNF-α-308AA in patients with colorectal cancer was significantly higher than that in healthy subjects (x2 =6.15, P<0.05, OR=2.08, 95%CI=1.17-3.71), while the frequency of IL-6-572CC in patients with colorectal cancer was significantly lower than that in healthy subjects (x2 =4.97, P<0.05, OR=0.73, 95%CI=0.55-0.96).The frequency of TNF-α-308AA in patients with colon cancer (OR=2.31, 95%CI=1.17-4.55), tubular adenocarcinoma (OR=2.32, 95%CI=1.28-4.21), high (OR=2.05, 95%CI=1.01-4.15) or moderately differentiated adenocarcinoma (OR=5.88, 95%CI=1.79-19.30) was significantly higher than that in healthy subjects.The levels of serum TNF-α in TNF-α-308AA carriers with colorectal cancer were significantly higher than those in TNF-α-308G carriers with colorectal cancer (t=2.13, P<0.05) as well as those in healthy TNF-α-308AA carriers (t=2.13, P<0.05).The levels of serum IL-6 in colorectal cancer group were significantly higher than those in control group (t=6.74, P<0.001).Conclusion The SNPs of TNF-α-308 and IL-6-572 are associated with the occurrence and development of colorectal cancer in Chinese Han people in Shandong province.

Objective To study the relationships of TGFβ1 (-509C/ T, +869T/ C) and TGFβR2 (-875 G/ A) single nucleotide polymorphisms (SNPs) with colorectal cancer (CRC) in Chinese Han popu-lation in Shandong. Methods TGFβ1 -509C/ T and +869T/ C SNPs in a total of 490 patients with CRC were detected using gene chip. TGFβR2 -875 SNPs was analyzed using PCR-RFLP. TGFβ1 concentrations in serum samples were measured by ELISA. Immunohistochemistry was used to detect the expression of TGFβR2. The relationships of TGFβ1 (-509C/ T, +869T/ C) and TGFβR2 (-875 G/ A) SNPs with CRC were analyzed through a case-control study. Chi-square test or t test was used for statistical analysis. Rela-tive risk was estimated by odds ratio (OR) and 95％ confidence interval (95％ CI). Results No signifi-cant difference in genotype or allele frequency at TGFβ1 -509 / +869 was found between patients with CRC and healthy subjects (P>0. 05). The frequencies of TGFβR2 -875GG genotype and -875G allele in pa-tients with CRC were significantly higher than those in healthy subjects (-875GG: χ2 = 4. 65, P = 0. 031, OR=1. 32, 95％ CI=1. 03-1. 71; -875G: χ2 =4. 95, P=0. 026, OR=1. 29, 95％ CI=1. 03-1. 61). Com-pare with the healthy control group, higher frequencies of TGFβR2 -875GG genotype and -875G allele were also detected in rectal cancer ( -875GG: P = 0. 04, OR = 1. 39, 95％ CI = 1. 02-1. 95 and -875G: P =0. 045, OR=1. 32, 95％ CI = 1. 01-1. 73), tubular adenocarcinoma ( -875GG: P = 0. 004, OR = 1. 51, 95％ CI=1. 14-2. 00 and -875G: P=0. 003, OR=1. 45, 95％ CI=1. 14-1. 85) and highly differentiated tu-bular adenocarcinoma (-875GG: P=0. 003, OR=1. 68, 95％ CI=1. 19-2. 38 and -875G: P=0. 002, OR=1. 62, 95％ CI=1. 18-2. 21) groups. The serum TGFβ1 levels in TGFβR2 -875G carriers with CRC were significantly higher than those in TGFβR2 -875AA carriers in both CRC (t= -3. 42, P<0. 05) and healthy control (t= -5. 09, P<0. 001) groups. TGFβR2 expression in -875G carriers with rectal cancer was signifi-cantly lower than that in -875AA carriers with rectal cancer (P=0. 047) and healthy subjects (P=0. 027).Conclusion TGFβR2 -875GG might be a potential risk factor for CRC in Chinese Han population in Shandong and TGFβR2 - 875G might be a risk factor for rectal cancer and highly differentiated tubular adenocarcinoma.