| Part one: to study IL-21in serum of colon carcinoma patients and its clinical significance objective: We analyzed the differences of IL-21 and IFNγin different sex, ages, pathologic stages, pathologic types and lymph node transmission by assaying its secretion level in serum of colon carcinoma patients. We further discussed the relationship between IL-21 and IFNγand the effect to progress of IL-21 under different clinical pathologic factors, in order to offer the theory basis for improving IL-21 level to inhibit growth of colon cancer cell in the forthcoming immunity gene therapy. Methods: 1 We randomly collected 40 cases of colon carcinoma patients who were operated in the Surgery Department of No.4 Hospital of Hebei Medical University during March 2004 to October 2004. We confirmed the diagnosis by pathologic examination and identified they were not treated and didn't use any immunity drugs. We collected 3ml vein blood on an empty stomach before three days of the operation. Draw 1ml serum and calculate the density of IL-21 and INF in the sample by ABC-ELISA. 2 We calculated the volume of the tumor by strictly measuring its length, width and height after sample resection. We dissected the regional lymph nodes carefully and examined them by pathologic method in order to calculate the rate of lymph node transmission. 3 We analyzed and statisticed data by t-test, chi-square test, correlation analysis and so on. Results: 1 After pathologic examination, 21 cases were no lymph node transmission, on the other hand, 19 cases were found lymph node transmission in different degree among 40 cases. We collected 361 lymph nodes in the 40 patients and found there were 86 lymph nodes transmission, the rate of transmission was 23.8%. The contents of IL-21 and IFN-γin the serum was 1879±212pg/ml, 17.48±4.14pg/ml individually in the no lymph node transmission group; The contents of IL-21and IFN-γin the serum was 1444±121pg/ml, 15.21±2.79pg/ml individually in the lymph node transmission group. By statistical analysis, there was significant difference of IL-21and IFN-γin the lymph node transmission group(P<0.05). 2 We divided 40 cases into 2 groups by median age. The detection value of IL-21and IFNγwas 2004±221pg/ml, 16.95±2.86pg/ml individually in the older group, while The detection value of IL-21and IFN-γwas 1228±141pg/ml, 15.97±3.51pg/ml individually in the younger group. The level of IL-21 in the older group was lower than that of in the younger group. By statistical analysis, there was significant difference in two groups(P<0.001). However, there was no significant difference of IFN-γin the two groups(P>0.05). 3 In the different sex groups, the level of IL-21 and IFN-γin the male colon carcinoma patients was 1647±253pg/ml, 16.60±3.28pg/ml individually, while the level of IL-21 and IFN-γin the female colon carcinoma patients was 1578±265pg/ml,IFN-γ16.28±3.88pg/ml individually. There were no significant difference in the two groups(P>0.05). 4 The content of IL-21 in DukesA stage,DukesB stage,DukesC stage and DukesD stage was 1371±116pg/ml, 1630±197pg/ml, 1913±117pg/ml, 1983±222pg/ml individually. The content of IFN-γin DukesA stage,DukesB stage,DukesC stage and DukesD stage was 19.55±2.06pg/ml, 17.48±2.47pg/ml, 15.93±2.43pg/ml, 14.83±3.13pg/ml individually. From the above, the level of IL-21 in the colon carcinoma patients increased gradually with the disease development. By statistical analysis, there was significant difference among the four groups(P<0.05). Although the content of IFN-γin the serum showed a decreasing tendency, there was significant difference between Dukes A stage and Dukes B stage(P<0.001). There was no significant difference among the other stages(P>0.05). 5 The density of the two cell factors showed no significant changes in different pathologic types. The content of IL-21 in the high differentiation adenocarcinoma, the medium differentiation adenocarcinoma, the low differentiation adenocarcinoma and mucous adenocarcinoma was 1654±295pg/ml, 1822±272pg/ml, 1738±281pg/ml,1739±308pg/ml individually, while The content of IFN-γin the high differentiation adenocarcinoma, the medium differentiation adenocarcinoma, the low differentiation adenocarcinoma and mucous adenocarcinoma was 16.95±2.55pg/ml, 16.16±2.59pg/ml, 18.88±2.44pg/ml, 14.4±3.18pg/ml individually. By statistical analysis, there was no significant difference of IL-21 and IFN-γin the groups(P>0.05). 6 We divided 40 cases into 4 groups. They were mucous membrane invaded, superficial muscular layer invaded, deep muscular layer invaded and peri-soft tissue invaded. The content of IL-21 and IFN-γin the four groups was 1421±155pg/ml, 17.05 ±2.06pg/ml, 1532±126pg/ml, 16.6±2.32pg/ml, 1725±201pg/ml, 16.67±2.47pg/ml, 1905±214pg/ml, 16.23±3.02pg/ml individually. With the increasement of invasion depth, the density of IL-21 increased. By statistical analysis, there was significant difference in the groups(P<0.05) except superficial muscular layer invaded group and deep muscular layer invaded group. There was no significant changes in the density of IFN-γ. There was significant difference between deep muscular layer invaded group and peri-soft tissue invaded group (P<0.05). 7 By statistical analysis, We found the relationship between IL-21 and IFN-γwas positive correlation(rIFN-γ=0.335). For the age factor, the level of IFN-γand age was positive correlation(rIFN-γ=0.251),while there was no positive correlation between IL-21 and age(rIL-21=0.096).The level of IL-21and different pathologic stage show positive correlation (rIL-21=0.243). The level of IFN-γand different pathologic stage show no significant positive correlation(rIFN-γ =-0.170). there was no positive correlation among IL-21, IFNγand different pathologic types(rIL-21=0.005,rIFN =-0.095).The invasion depth and IL-21 show positive correlation(rIL-21=0.2681). The invasion depth and IFN-γshow no positive correlation(rIFN-γ =-0.042). IL-21 , IFN-γand lymph node transmission show positive correlation ( rIL-21=0.238 , rIFN =0.304).The level of IL-21and the volume of tumor show positive correlation(rIL-21=0.283,),while The level of IFN-γand the volume of tumor show no positive correlation (rIFN-γ=-0.073,). Conclusions: Interleukin (IL)-21 was a recently described type â… cytokine which could kill some kinds of tumor cells by modulating B,T,and natural killer cell responses. It could promote the secretion of the IFN-γand yielded anti-tumor effect in coordination with the latter. The two factors showed positive correlation tightly in colon carcinoma patients. But with the increasement of age, the enlargement of tumor volume, deepen invasion, stage later and lymph node transmission, the level of serum IL-21 was not enough relatively. By using immunity technology to improve its secretion level, we could make it be a kind of effective method cure colon carcinoma. Part two: to study colon carcinoma cell transduced with IL-21 to inhibit the growth of tumor Objective: We proposed to develop a mouse model of metastasis of murine colon carcinoma cells(Colon26 cell) and develop a murine colon carcinoma cellline of metastasis that were transduced with IL-21 gene(Colon26/IL-21 cell). We examined the biological character and the effect on tumor cell proliferation and transmission in order to discover the mechanism of inhibiting tumor cell proliferation and lymph transmission by inducing the immunity of body. Methods: 1 Murine colonrectal carcinoma Colon26, a undifferentiated adenocarcinoma, was transplanted into subcutaneous tissue of homogenous Balb/c murine inner pod. After 25 breed days, popliteal fossa lymphonodus was obtained in asepsis condition, then granulated it into granule through a net, grounded tissue was developed in RMPI1640 medium supplemented with 10% fetal calf serum. Then monocelled clone of the metastatic tumor cell was obtained, further cellline grew well was studied by MTT. 2 Colon26M cellline that can express IL-21 gene, named Colon26M/IL-21, was obtained by the retrovirus vector LXSN which have carried IL-21 DNA through ecotropic ψ2 cell and amphotropic PA317 cell transfected conlon26 cell. 3 We analysed the gene expression, cell proliferation and MHC molecular changes of colon26M/IL-21. 4 Colon26M/IL-21 cellline was transplanted into murine single inner pod of each mouse as a experiment group, and Colon26M celline as a control, tumor growth of each transplanted murine pod was observed, andthe tumor volume was evaluated. 5 The production of IFN-γwas examined by ABC-ELISA method, stimulated the body to produce IFN-γand inhibit tumor to grow. Results: 1 After proliferation of single cell, we found the specific brown-yellow cell plasma in the colon26 cell. The cell was identified as high transmission carcinoma cell. 2 Establishment of murine colon carcinoma Colon26M celline that can express IL-21 gene:Colon26 celline that can express IL-21 gene was established by retrovirus vector LXSN which carried IL-21 cDNA,and it was named Colon26M/IL-21.Both bright 28sRNA bands of Colon26M/IL-21 and Colon26M were observed in gel scan showed that both general RNA had been isolated. It was demonstrated that the Colon26M/IL-21 celline can express IL-21mRNA by special DNA band on 434bp mark had seen in DNA products gel scan which had obtained from amplication by RT-PCR method. 3 In vitro, Colon26/IL-21 could proliferation normally. But compared with colon26, there was no change in proliferation ability. 4 We analyzed the expression of MHC I molecule on the colon26 cell by indirect immune Fluorescent assay. The results showed that MHC I molecule expression of Colon26M/IL-21 was higher than that of colon26. 5 The tumor formed in the left pad of experimental mouse fingers, the mean volume of tumors was 0.8 cm3, but the tumors gradually regressed, the mean volume was 0.22±0.10cm3; while the tumors of the control group still grew, the mean volume was 1.17±0.59cm3. There was significant difference between the two groups(P<0.05). 6 There was 1case of lymph node transmission in the experimental group and the rate of transmission was 7%. There were 6 cases of lymph node transmission in the control group and the rate of transmission was 40%. Slides were reexamined by CEA immunohistochemistry and its result were the same as above. There was different between the two groups. 7 We detected the secretion of Colon26/IL-21 by ELISA. The count was 179.67±11.32pg/ml, significantly higher than the content of Colon26, 32.32±6.42pg/ml(P<0.05). Conclusions: To dissociate from transmission lymph node in murine andcreate colon26 cell lines by colon assay, we transferred the IL-21 gene to colon cell by retrovirally transduced technique in order to induce murine to improve the content of INF-γ, increase the expression of MHC molecule and strengthen ability of CTL to inhibit the growth of colon tumor. On the other hand, the mechanism of specific immunity could effectively inhibit the lymph transmission of colon carcinoma. It was an important significance to clean the rest tumor cells in post-operation of colon carcinoma and avoid the transmission and recurrence of colon carcinoma. Part three: to study the anti-tumor effection of colon carcinoma cell vaccine by irradiating transduced IL-21 objective: In our experiment we want to culture the non-oncogenic colon 26/IL-21 cells vaccine by irradiating at isotope Co60 50Gy, study the vaccine's character non-Oncogenic of colon 26/IL-21 cells contrast with colon 26/IL-21 cells and it's antitumor effects.So we could provide the base theory in gene therapy of human colon carcinoma. Methods: 1Murine colon carcinoma cells(colon 26) that were retrovirally transduced with mouse IL-21 gene were irradiated at isotope Co60 50Gy,so we obtained the vaccine of irradiated colon 26/IL-21 cells. 2 The growth curve about vaccine of irradiated colon 26/IL-21 cells were draw by MTT method and compared the different of cells proliferation between anterior-irradiated and post-irradiated; The transcription of IL-21 gene after post-irradiated in different period were detected by RT-PCR method. 3 The changes about cell cycle and apoptosis rate between anterior-irradiated and post-irradiated were detected by FCM. 4 The changes that spleen cells ability of secreting IFN-γunder different stimulation were deteced by ELISA method. To examine vaccine's development and antitumor effects in mouse we inoculated the vaccine of irradiated colon 26/IL-21 cells in subcutaneour. Results: 1The multiplie time of Colon26/IL-21 cells is about 2.1 days. The cellproliferation colon 26/IL-21 cells that were gave a dose isotope Co60 50Gy .We culture the irradiated colon 26/IL-21 vaccine cells in vitro, the number of vaccine cells decrease 50% after 4 days, while the number ofvaccine cells decrease 30% after 7 days. 2 FCM analysis demonstrated that Cell cycle distribution and apoptosis have an obvious change between anterior-irradiated and post-irradiated: Vaccine of irradiated colon 26/IL-21 cells were induced accumulationin G1(79.421 ±4.860%) ,which was accompanied by decrease in the percentage of cell in S phase,with statistical significance of P<0.01.The apoptosis percentage of irradiated colon 26/IL-21 cells was 35.547±3.787% after culture 4 days later.It has an obvious change compare with anterior-irradiated 0.734±0.115% which has a statistical significance of (P<0.01). 3 The transcription of IL-21gene after post-irradiated in different period (1.3.5days)were detected by RT-PCR method, which indicated the transcription of IL-21 gene reduced gradually. 4 In vivo , we inoculated the vaccine of irradiated colon 26/IL-21 cells at a dose 1×106 in one pad of mouse finger, there was no tumor development after 7 days, observe continuously no tumor developed till 45 days. 5 In vitro, The changes that spleen cells ability of secreting IFN-γunder different stimulation were detected by ELISA method. The level of IFN-γthat spleen cells secreted was 23.767±4.123pg/ml when we culture the spleen cells and colon 26/IL-21 cells together, the level of IFN-γthat spleen cells secreted was 21.930±7.786pg/ml when we culture the spleen cells and irradiated colon 26 cells together, The level of IFN-γthat spleen cells secreted was 10.900 ±2.524pg/ml when we culture the spleen cells and irradiated colon 26 cells together. 6 We inoculated the colon 26 tumor cells at a dose 5×103 in each pad of all mouse finger. And then we continuously inoculated the vaccine of irradiated colon 26/IL-21 cells at a dose 1 ×105 in subcutaneour of experimental group every 4 days, repeat this for 5 times. All of the mice developed tumor at the pad after 7 days later. The volume of experimental group tumor developed to 7.963±1.115cm3 till 26 days later, compare with control group 8.270±1.372cm3, have no statistical significance(P>0.05). Secondly, we inoculated the colon 26 tumor cells at a dose 5×103 in each pad of all mouse finger. And then we continuously inoculated the vaccine of irradiated colon 26/IL-21 cells at a dose 2 ×106 in subcutaneour of... |
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