Changes Of Immune Cells And Its Association With HBV-DNA, HBeAg Tumor Stage In Patients With Primary Hepatocellular Carcinoma

Background and objectivePrimary hepatocellular carcinoma (Primary hepatic carcinoma) refers to the malignant tumor that is derived from the liver and HCC (Hepatocellular carcinoma), which accounts for more than 90% of Primary liver cancer. Its incidence of cancer reach the sixth among the tumor deseases all over the world, and the mortality of cancer related deaths is third. Early surgical excision is the treatment of primary liver cancer.However, because of its hidden, no obvious symptoms early, being in the middle-late when to see a doctor, the operation effect is far to being ideal. The 5 years of survival rate is only about 50%. Studies have confirmed the body’s immune system plays important role on the curative effect and prognosis of primary liver cancer. The immune system is a kind of physiological function of the body, which relies on this kind of feature recognition "self and " non-self" composition in order to effctively deal with the antigen from outside or produced by the human body damage cells and tumor cells. and consist of themmune defense and immune surveillance function. Immune cell is the basic unit of the immune system, mainly includeing:neutrophils, T lymphocytes and B lymphocytes and NK cells, etc. The series reaction of immune cells to antigen is called the immune response. Immunology in the early development of exogenous antigen specific immune refer to be the body immune reaction. Any internal and external environment of the physical and chemical factors stimulation can activate the body’s response to pursue benifits and avoid damage. Along with the development of immunology, in the middle of the 20th century, it was found that the immune system can identify the tumor and responds to its, and through the animal experiments proved that the existence of tumor antigen and the induction of immune response has antitumor effect. The status of the body’s immune function is closely related to the occurrence and development of malignant tumor. Under normal circumstances the body can be monitor and clearmutant cells or cancer cells, which can prevent the tumorigenesis desease. When the body’s immune function is weak, the variation and malignant cells can escape the immune system with a variety of ways of monitoring and the unlimited growth and lead to tumorigenesis. In the resistance of tumor immunity, cellular immunity, especially T lymphatic cell plays a key role. Current research has confirmed that the occurrence, development and prognosis of liver cancer are closely associated with the body’s immune status, and liver cell necrosis can induce specific T cell immune response. After the occurrence of tumor mainly through the body of T cell subsets of tumor cells in the immune damage, at the same time affect the total volume of the tumor and tumor stage. HBV-DNA (Hepatitis B virus get-quantitative DNA) is mainly used to valuate people infected with Hepatitis B virus in the body, to a certain extent, reflects the HBV replication activity HBV persistent infection is an important factor of HBV related liver cancer occurrence and development, and also closely related to the innate immune system. This study cells through the determination of hepatocellular carcinoma in patients with preoperative peripheral blood T lymphocyte subsets changes, To explore of primary hepatocellular carcinoma preoperative T cell immune function status, ratio of NK cells, neutrophils and lymphocytes (neutrophil to lymphocyte thewire, NLR) related influencing factors.Method1 Subjects 45 patients with primary HCC in nanfang hospital department of hepatobiliary surgery in Guangzhou from October 2014 to November 2015 postoperative pathology were recruited as experimental groupaccording to the inclusion criteria:(1) patients with HBsAg positive; (2) liver Child-Pugh class for grade A or B; (3) no preoperative intervention, radiofrequency ablation,chemical treatment and radiation treatment; (4) preoperative eliminate disease such as infection, blood system diseases; (5) For radical primary HCC resection surgery, postoperative pathological cut edge negative. Including 30 cases of male patients, 15cases were female patients, aged 37 to 63 years old, the median age 50; Control group for the same time nanfang hospital physical examination center normal adults, among them 29 cases were male, female 16 cases, age 27 and 67 years of age, the median age of 42. Collection including age, gender, preoperative serum AFP (AFP) level, HBV-DNA ration, HBeAg, preoperative peripheral blood T lymphocyte subsets in one weeks, blood routine, liver function, blood coagulation function, such as inspection result, imaging data, including tumor size, location, number, ascites, liver cirrhosis, and so on and so forth. Postoperative pathological data including pathological type, tumor diameter, the degree of vascular invasion, specimens cut edge and liver Child-Pugh, classification of clinical data.2, study method In all cases of control group and control group in a week in a medical extract on the day of early morning on an empty stomach 10 mL peripheral venous blood were analyzed. Heparin anticoagulant tube collection, collection within 6 hour after processing.100 ul peripheral blood from each, respectively, to join "CD4-FITC, CD8-PE, CD3-PerCP", or " CD8-FITC, CD28-PE", " CD25-PE, CD4-FITC" or "CD3-FITC、CD16-PE、CD56-PE" tag, flow cytometry detection line CD3+T cells and CD4+T cells, CD8+T cells and CD4+ /CD8+ratio, CD8+CD28-T cells and CD8+CD28+T cells,CD4+CD25+T cells and NK cells frequency.The experimental group in peripheral blood loss of cytology, the specimen on the blood day processing. Neutrophil to lymphocyte ratio(NLR) and total tumor volume(TTV) were calculated according to the clinical data. Tumor volume calculation method can be reference ball volume formula:tumor size (cm)=4/3x3.14×r3 after the largest radius (r=measurable tumor nodules). TTV volume sum equal to the measurable lesions. Tumor radius by preoperative imaging examination results can be obtained. NLR according to patients with preoperative peripheral blood routine inspection result within 1 week of neutrophils and lymphocytes value ratio.3, Statistical methods By SPSS 19.0 software for statistical analysis of data. The form of a continuous variable to mean+/-standard deviation, comparison between the two groups by using two independent sample t test. Bilateral inspection P value is less than 0.05 think have statistical significance, correlation analysis using Pearson correlation analysis.Conclusions3.1 Differences in the levels of immune cells between liver cancer group and the control group:Patients with primary hepatocellular carcinoma CD3+ T cells and CD4+T cell frequency and ratio of CD4+/CD8+T cells significantly lower than the control group, statistically significant difference between the two groups,, the P values were 0.015,<0.001,< 0.001, CD8+T cells and primary hepatocellular carcinoma group value is significantly higher than normal group, statistically significant difference between the two groups (P=0.035), patients with liver cancer, T cells in the body and the group changes. Further analysis, CD8+CD28- and the frequency of CD4+CD25+cells in liver cancer group is higher than the control group, the difference is statistically significant P value were 0.008,0.041, and CD8+ CD28+T cells, frequency no statistical differences between the two groups (P= 0.166), the weak T cells in patients with liver cancer, an increase in frequency.3.2 Barcelona stage (BCLC) in patients with liver cancer in different between immune cell difference:BCLC 0 period of 10 patients with this topic, A period of 16 cases, B period (19 cases), CD3+T cells and CD4+T cells and CD8+CD28+T cells frequency respectively in each stage did not differ significantly between (Kruskal Wallis H test, P> 0.05). CD8+T cells was statistically difference between frequency in the three stages (the Mann-Whitney U test, P=0.042), with the increase of the level, frequency of CD8+T cells has a tendency to rise, in the mid-term B is higher than 0 (P= 0.035). Ratio of CD4+/CD8+T cells in the three stages between statistically significant (P=0.043), with the increase of the level, the ratio of CD4+/CD8+T has a tendency to reduce, in the mid-term below 0 B period (P=0.032). Frequency of CD8+CD28-T cells and CD4+CD25+T cells there are statistical differences between three stages respectively (P=0.045 and P= 0.041), with the increase of Barcelona stage level, both has a tendency to rise, in the mid-term B were higher than 0. The frequency of NK cells and NLR between each installment no statistics.3.3 Primary hepatocellular carcinoma (HCC) in patients with immune cells and the relationship between the HBV DNA:CD3+T cells and CD4+T cells and CD8+T cells and CD4+/CD8+ratio, CD8+CD28-T cells and CD8+CD28+T cells and CD4+CD25+T cells, NLR frequency and the frequency of NK cells in HBV DNA respectively< 1 x 103 copies/mL and HBV DNA> 1 x 103 copies/mL no statistical difference (P> 0.05) between。3.4 The patients with primary hepatocellular carcinoma(HCC)with HBeAg immune cells:CD3+T cells and CD4+T cells,CD8+T cells and Ratio of CD4+/CD8+T cells,CD8+CD28一T cells and CD8+CD28+ T cells and CD4+CD25+T cells NLR frequency and the frequency of NK cells in HBeAg+and HBeAg-no statistical difference between(P>0.05).3.5 All patients with primary hepatocellular carcinoma(HCC)T cells relations with tumor size,on the one hand,we will single diameter of liver cancer<5 cm patients divided into for small hepatocellular carcinoma group(small),multiple liver cancer or single hepatocellular careinoma diameter> 5 cm for patients with large liver cancer group(huge),the result shows；the CD3+T cells and CD4+T cells and CD8+T cells and CD4+CD25+T cells and NK cells in frequency between the size 0f liver cancer group have no statistical difference(P>0.05),Ratio of CD4+ /CD8+T cells in a large group of liver cancer than small liver cancer group increased significantly lower(P=0.002),similarly, CD8+CD28-T cells in a large grobp of liver cancer than small liver cancer group increased significantly(P= 0.009),NLR(FIG.3-4 I)in a large group of liver cancer is higher than that of small liver cancer group.Frequence,on the other hand,we will all the cells and CD4+/ CD8+T cell ratio relation with TTV correlation annlysis,the results showed the CD4+／CD8+T cell ratio was positively related with TTV(P=0.001),and CD3 +T cells and CD4+T cells and CD8+T cells,CD8+CD28-T cells and CD8+ CD28+T cells and CD4+CD25+T cells frequency has no obvious correlation(P >0.05,figure 3-2)。ConclusionsPrimary liver cancer patients with T cell subsets in the body is different from normal,peripheral blood CD3+T cells and CD4+T cells lower than normal,and CD8+T cells,CD8+CD28-T cells and CD4+CD25+T cells subgroup level is significantly higher than normal; Cancer of the liver, the progress of tumor may and CD8+CD28-T cells and CD4+CD25+T cells subgroup level, the higher the more relevant. The two groups of regulatory T cells may participate in the occurrence and development of liver cancer, may be related to the curative effect of hepatocellular carcinoma.