| The incidence and fatality rate of malignancy tumor is very high, and it has become the number one killer of human disease around the world. However, the cure rate of three conventional therap ies(surgery, chemotherapy and radiotherapy) for tumor was very low, and there were a lot of side effects.In recent years, tumor biologicaland cell therapy for tumor treatment brings us new hope. As our best knowledge, human tumor necrosis factor α(hTNFα) has strong effects on killing or inhibiting the growth of tumor cells, and it has been used in clinical treatment of some types of tumor. Because the hTNFα has a short half- life, the drug concentration was low when arriving in tumor tissue after intravenous systemic. Furthermore, the effect time of drug was short, so it was difficult to achieve the satisfactory effect on tumor inhibition. At mean time, the use of large doses could cause serious systemic adverse reactions, such as chills, fever and shock. As a result, it was applied in clinical with greatly limitation. Our team previously has used the embryonic kidney cells as the carrier, and we established the hTNFα/293 cells, which could released hTNFα sustainability. By using the APA microencapsulate the cells, the APA-hTNFα/293 cell microencapsulations implanted inside and around the tumor tissue in tumor-burdened nude mouse. Our results showed that hTNFα was released through the cells in the microencapsulations sustainability in tumor tissue and local, with the functions of immune isolation the APA- hTNFα/293 cell, the microencapsulations could inhibit the tumor growth in an effective way. It also could improve the concentration of hTNFα and prolonged the action time in tumor tissue local, reduced the systemic adverse reactions, and had been approved to inhibit the tumor cell growth effectively.As a new method of tumor inhabiting, we need to study further to prove whether it can be played a role in tumor growth inhibition by carrying in a variety of carrier cells through the tumor growth inhibition experiments.This research used CHO cells(C hinese hamster ovary cells) as a carrier, the application of liposome transfection technique, and established the hTNFα/CHO engineered cells. By using the APA microencapsulated the engineered cells, preparation of APA- hTNFα/CHO cells microencapsulations, implanted in and around tumor-burdened nude tumor tissues, overcome immune rejection of allogeneic cells, to provide continuous hTNFα which released by micro biological pump function of hTNFα/CHO cells, maintained the high concentration hTNFα around the tumor local, low concentration hTNFα in body, in order to give full play to the functions of hTNFα of tumor growth inhibition, and reduced systemic adverse reactions. The treatment of malignant tumor is dedicated to provide a new biological treatment method. Objective:We establish the hTNFα/CHO engineered cells; preparation of APA-hTNFα/CHO cells microencapsulations. In vitro and vivo, to known this cell microencapsulations was inhibited the tumor cell growth and the side effects of the host. Content:Used liposome transfection technique, we established the hTNFα/CHO engineered cells; In vitro co-culture experiments showed that the cell can express hTNFα gene and sustainable release hTNFα; Used ELISA method to detect the hTNFα protein content in the cell culture. Preparation of the APA- /CHO cells microencapsulations. Co-culture the APA- hTNFα/CHO cells microencapsulations and 9 kinds of human tumor cells in vitro to observe the tumor cell growth inhibition effect of APA cell microencapsulations. The APA-hTNFα/CHO cells microencapsulations implants tumor-burdened(LO VO of human colon cancer, HEPG2 of human liver cancer) nude mouses in and around its tumor tissue, observe its tumor growth inhibition effect, and influence on the host weight and survival. Method:1. The hTNFα/CHO engineered cell lines were established: we synthesized and amplified of hTNFα purpose gene by PCR technology, transferred gene by plasmid restriction analysis and recombinant technology into the plasmid GV141 and tested purposes gene integration level. we transducted gene recombinant plasmid GV141 into CHO cells by Lipofectamine 2000, established engineering cells hTNFα/CHO cell line by G418 resistance selection. we extracted the total RNA from hTNFα/CHO cell, detected hTNFα/C HO cells of exogenous hTNFα genes by PT-PCR method and hTNFα protein in normal culture hTNFα/CHO cell culture supernatant by protein ELISA kit; we observed growth condition of hTNFα/CHO cells and CHO cells by inverted microscope, measured cell growth curve of them by MTT method and contrasted the change.2. The tumor inhibition effect was observed by co-culture tumor cells and APA-hTNFα/CHO cell microencapsulations: the APA capsulated hTNFα/CHO cells and 0/CHO(transfer plasmid GV141 without hTNFα gene) to make APA-hTNFα/CHO and APA-0/CHO cells microencapsulations. We observed them form and the growth situation with inverted optical microscope. The APA-hTNFα/CHO cells microencapsulations co-cultured with nine different human tumor cells(liver cancer cells HEPG2, colon cancer cell LOVO, cervical cancer cells HELA, lung cancer cell H460, breast cancer cells MCF-7, bladder cancer cell T24, breast cancer cells T47 D, lung adenocarcinoma cells A549, kidney cancer cells ACHN). Set the normal growth of tumor cells to blank control; hTNFα solution as the positive control; Preparation of APA-0/CHO cells microencapsulation as the negative control; Different doses(low dose:50 medium dose:100 high dose:200)of the APA-hTNFα/CHO cell micro- encapsulations as test group, use MTT method, we checked the growth condition in normal culture of human tumor cells and drew the tumor cell proliferation curve, observed the APA-hTNFα/C HO cell microencapsulations inhibition of tumor cell growth to nine different human tumor cells. Data was analyzed by T test with SPSS19.0 software.3. The tumor-burdened nude mouse tumor tissue inhibition was observed by implanted APA-hTNFα/CHO cell microencapsulations: Referenced the results in above, we selected two human tumor cell to make tumor-burdened nude mouse transplantation tumor model, preparation of APA- hTNFα/CHO cell microencapsulations was implanted in and around the tumor-burdened nude mouse tumor tissue. Injected with normal saline group for blank control; Injected hTNFα solution as the positive control; Injected APA-0/CHO cell microencapsulations as the negative control; Injected different doses of the APA-hTNFα/CHO cell microencapsulations for the test group. Every 3 days after implantation, we observed the tumor-burdened nude mouse survival state, weighed its weight and measured its length and short diameter of tumor tissue. 21 days after implantation, we executed the tumor-burdened nude mouse with spinal dislocation method, separated of a tumor-burdened nude mouse tumor tissue, weighed the weight of tumor tissue. Data was analyzed by ANOVA with SPSS19.0 software. Result:1. Established hTNFα/CHO engineered cells and hTNFα protein secretion of verification:PCR gene tests showed recombinant plasmid contained hTNFα gene; Positive cloning genes and hTNFα gene sequencing comparison results showed that the integrated hTNFα gene sequence is no significant difference; The RT-PCR results showed positive control GAPDH gene electrophoresis banding display is normal, the RT-PCR detection system showed normal. The 4 in 5 transfected cells strains containing hTNFα gene, which the no.1 and no.2 electrophoresis banding is strong positive. Select no.1 hTNFα/C HO cell strains to continue the follow-up research. Measured cell culture supernatant with ELISA method. With the result above, we can know that hTNFα/CHO cells contain hTNFα purpose gene, it can encode hTNFα protein and release outside the cell continuing. Then measured OD by MTT method to draw hTNFα/CHO cell and CHO cells growth curve, we found its no appear the apparent change.2. The inhibition effect of APA- hTNFα/CHO cell microencapsulations inhibiting tumor cell growth co-culture with tumor cells.⑴We can see with inverted optical microscope that APA- hTNFα/CHO cell microencapsulations are globular, and hTNFα/CHO cells distribute in APA microencapsulations uniformly, 3 days later hTNFα/CHO cells were growth in dough.⑵Compared with blank control group, APA-0/CHO cell microencapsulations group had no inhibitory effect on the growth of 9 kinds of tumor cells(P>0.05); Positive drug hTNFα group had significant inhibitory effect in addition to T47 D and HELA of 9 kinds of tumor cells(P<0.01), it had no inhibitory effect in T47 D and HELA cells.⑶ Compared with blank control group, each dose APA- hTNFα/CHO cell microencapsulations group had significant inhibitory effect in addition to T47 D and HELA of 9 kinds of tumor cells(P<0.01), medium dose of APA- hTNFα/CHO cell microencapsulations group had inhibitory effect in T47 D cells(P<0.05); High dose of APA-hTNFα/CHO cell microencapsulations group had significant inhibitory effect in 47 D and HELA cells(P<0.01); Most of APA-hTNFα/CHO cell microencapsulations had stronger inhibitory effect than positive drug hTNFα group(P<0.05); Part of APA-hTNFα/CHO cell microencapsulation had clear dose-response relationship.3. The inhibition effect of APA- hTNFα/CHO cell microencapsulation implants in nude mouse transplantation tumor.⑴ The inhibition effect of APA- hTNFα/CHO cell microencapsulation implants in LOVO tumor-burdened transplantation tumor nude mouse.The implanted APA-hTNFα/C HO cell microencapsulations of LOVO tumor-burdened transplantation tumor nude mouse weight had no effect(P>0.05), 21 days later, all nude mouse survived; compared with blank control group, positive drug hTNFα groups and high dose of APA-hTNFα/CHO cell microencapsulations groups had inhibitory effect in tumor tissue weight(P<0.05); The tumor volumes inhibition rate of High, medium and low dose APA-hTNFα/CHO cell microencapsulations groups were 31.9%, 29.1% and 14.7% respectively; Tumor weight inhibition rate were 54.8%, 46.2% and 54.8% respectively. Positive drug hTNFα group to tumor volumes inhibition rate was 48.9%, tumor weight inhibition rate was 63.5%. APA-0/CHO cell microencapsulations group to tumor volume inhibition rate was-21.8%; the tumor weight inhibition rate was 3.1%, it had no inhibitory effect.⑵ The inhibition effect of APA-hTNFα/CHO cell microencapsulation implants in HEPG2 tumor-burdened transplantation tumor nude mouse.The implanted APA- hTNFα/CHO cell microencapsulations of HEPG2 tumor-burdened transplantation tumor nude mouse weight had no effect(P>0.05), 21 days later, all nude mouse survived; compared with blank control group, all dose of APA-hTNFα/CHO cell microencapsulations groups had no inhibitory effect in tumor growth(P>0.05); The tumor volumes inhibition rate of high, medium and low dose APA-hTNFα/CHO cell microencapsulations groups were 14.6%, 7.9% and 7.6% respectively; Tumor weight inhibition rate were 7.5%, 3.2% and 12.7% respectively; Positive drug hTNFα group to tumor volumes inhibition rate was 45.3%, tumor weight inhibition rate was 45.4%. APA-0/CHO cell microencapsulations group tumor volumes inhibition rate was-35.6%, the tumor weight inhibition rate was-32.2%,it had no inhibition effect.Conclusions:1. The hTNFα/CHO cell line was established successful, it can stable genetic exogenous hTNFα gene, and through the prolife ration and metabolism, hTNFα gene can be expressed and released hTNFα protein out of the cell.2. APA-hTNFα/CHO cell microencapsulations group had inhibitory effect in 9 kinds of tumor cells,part of it had clear dose-response relationship. The hTNFα/CHO cells can release hTNFα to culture medium, and inhibited the growth of the tumor cells.We choose HEPG2 and LO VO cells into the next experiment.3. APA-hTNFα/CHO cell microencapsulations had significant inhibitory effect in LOVO tumor-burdened nude mouse and had inhibitory effect trend in HEPG2 tumor-burdened nude mouse; it had no effect in nude mouse survival. APA-hTNFα-/CHO cell microencapsulations released hTNFα to tumor tissues and inhibited tumor growth, especially in LOVO tumor-burdened nude mouse.The hTNFα/CHO cell line was established, under the APA microencapsulation package, through the vitro and vivo experiments proved that it had inhibitory effect in variety kinds of tumor cells. A further indicated that APA-hTNFα/X cell microencapsulation was expected to become new therapy products for the treatment of malignant tumor. it could overcome the shortcomings of allogene cell immune rejection problem and solved the problem of hTNFα clinical application, genetic engineering cells released sustaining of hTNFα as a micro biological pump to tumor local and lower concentration hTNFα in body, it could play the role as tumor inhibitory effect better, reduced systemic adverse reactions and provided a new effective method of adjuvant therapy for malignant tumor which difficult to cure. |
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