The Immunological Mechanism Of Ganzheng Solution For Hepatocellular Carcinoma

Objective Flow cytometry and ELISA were applied to investigate the change of immunological function in patients with hepatocellular carcinoma (HCC) and the effect of Ganzheng solution on immunological function in the patients with HCC. It is also explored how the serum containing Ganzheng solution affects the hepatocarcinoma cell proliferation, apoptosis, growing cycle and the expression of vascular endothelial growth factor mRNA (VEGF mRNA). We try to clarify the therapeutic mechanism of Ganzheng solution on patients with HCC by assessing its effect on immunology and biological behavior of hepatocarcinoma cell.Methods1. Subjects: 14 patients with HCC in the period of Ⅱb or Ⅲ phase were enrolled to this study from May to October in 2002. There is no further transfer, no opportunity of operation and no intervention of therapy in this group of patients. The grade of liver function was in Child-Pugh B (consisting with the revised diagnostic criteria and clinical stage criteria in the fourth National Hepatocarcinoma Academy). 30 healthy volunteers were chosen as control group. Five samples were randomly drawn from each group and everyone took the Ganzheng solution 40ml per day for 8 weeks. The blood was drawn for 10 ml from all the subjects.2. Test of the T cell subpopulations and NK cells: Lymphocyte groups and NK cells were defined by flow cytometer trischrom and recorded with ELITE4.5 software. After cell culture and staining, lymphocyte groups were decided in the FS and SS scatter diagram with flow cytometer trischrom investigation. According to the expression of IL-4-pe and INF- γ -fitc in the CD4 positive cells, Th1 and Th2 subgroups were determined and recorded.3. Detect the concentration of the cytokines in the serum: The concentration of cytokines in the serum was detected by the ELISA method.4. Observe the effect of the Ganzheng Solution on proliferation, apoptosis and growing cycle of SMMC-7721 Cells with the method of MTT, the technology of fluorescence microscopy and flow cytometer trischrom: After the cells had been cultivated for 24 or 48 hours, the inhibited effect of the Ganzheng solution on the proliferation of SMMC-7721 was determined by MTT chromatometry. The growth inhibition ratio (IR) was calculated according to the OD. After the observation of the cell division and apoptosis under the fluorescence microscope, the pictures were taken with the CCD camera. The cell growing cycle and apoptosis rate were detected with flow cytometry. The data was analyzed with MOFLB LT for Mac V2.0 software and at the same time the proliferation index (PI) was calculated.5. The effect of Ganzheng solution on the expression of vascular endothelium growth factor (VEGF) of SMMC-7221 cells: The cells were cultivated in the serum with and without drug for 48 hours before collected. RNA was drawn, followed by c-DNA collection with reverse transcription and amplified with the PCR equipment. The reacting product was added into 2% agarose for electrophoresis. The average gray scale of the electrophoresis band was detected in the result of the calculation of the ratio of the average gray scale of the VEGF and 0-actin band. The pair t-test was used in the statistics analysis.Results1. The difference of the T cell subgroups and NK cells between 14 patients with HCC and 30 volunteers: A. The mV of CD4+ in the patients with HCC was decreased obviously compared with that of the control group and so it was with CD4+/CD8+. Meanwhile, the mV of CD8+ is far higher than that of the control group. The distinction in statistics was significant, p<0.01. B: The mV of CD8+CD28" was significantly increased, p<0.01, while the mV of CD8+CD28+ (CTL) was decreased, p<0.01. C. The mV of CD3"CD56+(NK) of HCC was decreased, p<0.05, the same with CD3+CD56', but CD3+CD56+(NKT) was higher than that of the control group, p<0.05. D: Thl was decreased apparently, while Th2 was higher, and therefore Thl/Th2 wasdecreased, p<0.05.2. The difference of T cell subgroups and NK cells between 5 healthy persons and 5 patients with HCC before and after the solution-taking: A. Whoever they were, the healthy volunteers or the patients, the change of CD3+ could be statistically ignored. CD4+ was also subtle in the volunteers, p=0.493 while CD4+ was significantly increased among the patients with HCC after the medicine was taken, p<0.05; CD8+ and CD4+/CD8+ of the healthy volunteers were of no statistical difference, in contrast with that CD8+ of the patients HCC was decreased while CD4+/CD8+ was increased, but in statistics, the difference was subtle, p=0.183and p=0.124 because the samples were limited. B. CD8+CD28', CD8+CD28+of the healthy volunteers were the same before and after the medicine, CD8+CD28"of the patient was decreased while CD8+CD28+was reversed, p<0.05. C. CD3+CD56" of the healthy volunteers was higher than before,p<0.01, while CD3+CD56"of HCC was decreased, but there was no statistical difference, p>0.05. CD3+CD56+ of HCC was higher than before, p<0.05. CD3+CD56+ and CD3CD56+(NK) were of no obvious change among the healthy volunteers and it was the same with CD3'CD56+ (NK) in HCC, all of which could be statistically ignored. D. Among the healthy volunteers before and after the medicine was taken, there was no apparent change of the mV of Thl, Th2 and Thl/Th2 which was of no statistical value, while that of the patients with HCC Thl was higher afterward, p<0.01; Th2 was lower, p<0.05; Thl/Th2 was higher, which didn't produce statistical difference.3. The contrast of the cytokines between 14 patients and the control group of 30 volunteers: TNF-a and IL-10 of the patients with HCC were higher, p<0.05; IL-2 was lower, p<0.05.4. The change of the cytokines in both the healthy volunteers and the patients with HCC after the medicine: A. TNF-a, IL-2 and IL-10 of the healthy volunteers persons were not remarkably changed after the medicine which were not statistically different, p>0.05. B.TNF-a and IL-10 were all decreased, but IL-2 was significantly increased, p<0.05.5. The inhibiting effect of the serum containing drug on the growth of the hepatocarcinoma cells: The proliferation of SMMC-7721 cells which grew in the serum containing Ganzheng solution from the patients for 24 hours was inhibited. The inhibiting ratio was 45.00%, the value of the OD was 0.83±0.25 and 0.47±0.22 respectively before and after the medicine, p<0.01. The inhibiting ratio was 4.58% when SMMC-7721 cells grew in the serum containing Ganzheng solution from the healthy people; there was no significant difference in the value of the OD. The inhibiting effect of the serum containing drug from the healthy people on the hepatocarcinoma cells was much lower than that of the serum containing drug from patients. After the SMMC-7721 growing in the serum containing drug for 48h, the inhibiting ratio was 39.32% in patients and the value of the OD was 0.73±0.10 and 0.45±0.14 respectively before and after the medicine, p<0.01.The inhibiting ratio was 7.70% for the healthy people. Even though it was much lower than that for the patients, the inhibiting effect of the serum after the drug taking was stronger than that of the serum containing no drugs.6. Observe the effect of the Ganzheng solution on the proliferation and apoptosis of the hepatocarcinoma cells with the fluorescence microscope: A. Observation showed that many of the SMMC-7221 cells growing in the serum without Ganzheng solution were in the division period. After living in the serum containing Ganzheng solution for some time, the apoptosis rate of the SMMC-7721 cells increased significantly. As a result, out came many apoptosis cells, whose nucleoli had been disrupted and whose inspissated nucleoplasm scattered in the cytoplasm. B. The serum containing Ganzheng solution from healthy people in which the hepatocarcinoma cells were growing for 24h had less impact on the growing cycle. When the SMMC-7721 cells grew in the serum containing Ganzheng solution from patients for 24h, the number of the cells in the division period had been decreased sharply, and thus the division was under control.7. The effect of serum containing Ganzheng solution on the apoptosis and cell cycle of the hepatocarcinoma cells with flow cytometer : A. The resultimplied that the apoptosis ratio of SMMC-7721 cells increased both in the serum of the healthy people and of the patients after the medicine, and there was much statistical difference if compared. In the patients, the apoptosis ratio was 1.25±0.37 and 2.30±0.48 respectively before and after the medicine. After the medicine was taken, the apoptosis ratio of the cells in the serum of the patients was higher than that of the healthy people, but the number of the samples limited the statistical analysis. B. The influence of serum containing Ganzheng solution on the cell growing cycle of SMMC-7721 cells was not sharp in the healthy people in the stage from GO to Gl, of S and G2, p>0.05,and PI remained. C. Comparing the cells growing in the serum containing drug from the patients with those in the serum without drug from patients, we found that the percentage of the cells in the GO to Gl phase was greatly increased in the serum containing drug, p<0.05,while that of the cells in the S phase changed little, p>0.05. The percentage of the cells in the G2 phase decreased obviously, p<0.01, and the same thing happened to the proliferation index (PI), p<0.05. As a result, Ganzheng solution blocked the cell cycle of the hepatocarcinoma cells in the previous phase of the replication ofDNA.8. The result of RT-PCR production electrophoresis: VEGF (411bp) band was amplified after the healthy people's serum with and without drug was put into the bottles with the SMMC-7721 cells, but the light of the band changed little. The amplified band of P-actin also appeared at the corresponding site, and there was no significant difference of the gray scale value of VEGF/p-actin in healthy people before and after the medicine, (p>0.05). VEGF (411bp) band was amplified in the patients' serum with or without drug, there was significant difference of the gray scale value of VEGF/(3-actin in patients' serum with and without drug, p<0.01.543bp band appeared only in the serum without drug of only 2 patient and the band darkened after the drug taking.Conclusions1. The results above indicate that the immune functions were inhibited in...