| Objectives: Salivary gland tumors, as the second position of oral tumors, is one of the most common neoplasms in the oral maxillofacial regions, which is only less than oral cancer. Because salivary tumors are complex in the structure and pathologic type, most of the salivary tumors have potential malignancy. In addition, salivary tumors involve the oral maxillofacial regions and damage the facial features. So, it is a heat spot for research. Salivary pleomorphic adenoma (SPA) is the highest incidence of salivary tumors. It has the feature of border tumor and easy to relapse and implant. It is difficult to resect the relapse tumor surgically. Therefore, it is imperative to find a perfect auxiliary therapeutic treatment. It is not a clear realization about the pathogenesis of salivary pleomorphic adenoma. Along with the advance of the study, some scientists began to investigate the mechanisms of tumorigenesis. More documents have shown the fact that tumorigenesis is a polygenic disease involved in gene mutations which cause the hyperplasia of cell clone. The studies showed that during the pathogenesis of salivary pleomorphic adenoma, all genes of p53,ras,c-myc,c-erbB-2 had modified. So that, a theory has been put forward that salivary pleomorphic adenoma is a polygenic mutation disease. The present study is to solve whether the gene mutations in the tumorigenesis could be repaired or replaced, these data will be contribute to prevent and treat the tumor recurrence clinically. As a new method for tumor therapy, it has a longer development in gene theary. Current researches about gene therapy on animal models loaded various tumors have been reported. Hamada (1996) transduced cervix cancer cells with antisense RNA recommend adenovirus vector (Ad5CMV-HPV16AS) of human papillomavirus (HPV) E6 and E7 gene, when the cancer cells infected by HPV16 were transduced with Ad5CMV-HPV16AS, the proliferation of them was depressed. Swell (1997) treated epidermoid carcinoma with Herpes simplex virus-thymidine kinase (HSV-TK) gene by adenovirus vector in vivo, then injected with ganciclovir (GCV, a substrate of HSV-TK), the tumor becomes extinction. O'Malley(1998)treated murine salivary carcinoma with tyrosine kinase (TK) and murine interleukin (mIL-2) by adenovirus vector and got obvious effects. Drozik (2000) observed the independent and union treated effects of HSV-TK and IL-12 on hepatocellular carcinoma. They found that it could depress the proliferation of carcinoma and improve survival rate by union treation. Sun Chunxiao treated the adenoid cystic carcinoma by suicide gene and found that the suicide gene could effectively depress the small tumor. Although some advances in both tumor diagnosis and therapy come true, but the total effect is not satisfied, especially to the tumor metastasis and recurrenc. Recent reports about salivary tumors were only limited in salivary carcinoma and adenoid cystic carcinoma, there is no any report in salivary pleomorphic adenoma. The suicide gene treatment also named virus directed enzyme prodrug therapy (VDEPT). The theory of this method is that the tumor cells transduced with suicide gene could metabolize the atoxic prodrug into poison, which would depress the DNA synthesis and kill or wound the tumor cells. The immunity factor is one of the cellular regulator agents, which could enhance the body immunological function and enlarge the body immunologic reaction. If two pathways are combined, they will get the maximum kill effect on tumor cells. This study is to observe the depressing effects on pleomorphic adenoma by union treated with HSV-TK and IL-2 adenovirus vector. The results would establish the groundwork for the future clinical studies and applies, and provide some basic theory and experimental data for the tumor treatment. The present study was to devided into three parts:(1)Primary cellculture of salivary pleomorphic adenomas and identification;(2)The relationship between the gene therapy and ploid distribution of salivary pleomorphic adenomas;(3)The effects of HSV-tk suicide gene and IL-2 gene on salivary pleomorphic adenoma cells. Methods 1 Primary cell culture of salivary pleomorphic adenomas and identification Under the condition of sterile, the excisional tumour tissue was immediately put in culture medium containing 1000 U/ml penicillin and 500μμg/ml streptomycin, soaked for 15 min at room temperature to take off normal tissue,necrotic tissue,blood clot as well as envelope. Washed with PBS, then washed with RPMI-1640 supplement in 20 % hyclone, discarded supra suspension, sheared the tissues into about 1mm3 broken bits, the tissues were seeded in cultured flask and were divergenced to 2030 bits , added into culture medium , incubated at 37℃in 5% CO2 gas incubator for 3 days, observed under an inverted microscope and refreshed with culture medium. With the cells developed and populated, they were recorded by photogram timely. In addition, fibroblast cells were taken off by anchoring method repeatedly (2-3 times), and the pure tumour cells were left by this way. When the culture cells reached 80% confluency, the primary cultured cells were digested and passaged by using digestive juice containing 0.25% pancreatin and 0.02% EDTA (1:1). The culture medium of RPMI-1640 was supplemented by 1ng/ml epidermal growth factor and 5μg/ml transferrin to foster the tumor cells. When the cells grew well, counted and inocubated in 24 well plates at a density of 2×105/ml per well, added into equal quantity of culture medium, counted cells number in every 3 wells each day , together 8 days. The mean value was calculated and the diagram of curves was made to observe the viability of cells. Further, inverted microscope observation, histological staining and cytochemical staining, immunocytochemical staining and electronic microscope observation were used to assay the feature of tumor cells.2 The relationship between the gene therapy and ploid distribution in salivary pleomorphic adenomas Fifty samples of salivary pleomorphic adenomas were enrolled from paraffin sections kept in pathological archives. Ten normal salivary gland tissues were used as control. And another two fresh samples resected surgically were prepared. All of the paraffin samples were sliced and re-examined by two experience pathologic doctors to get correct diagnoses. Fifty patients with pleomorphic adenoma, 20 were males and 30 were females. Aged from 15 to 72 years old, and the average age was 40.1. The disease course was from 1 month to 22 years, and the average course was 3 years. All of the patients were undergone generally clinical examination before surgery. Referring to HE sections,each paraffin sample was sliced to obtain tissue section of 40μm, which was used to prepare single cell suspension. PI was inserted into the DNA fluorescence staining. The samples were analyzed by flow cytometer. The excitation light source was argon ion laser of 15mW, and the wavelength was 488nm. The DNA cell cycle was analyzed by Muticycle AV software. Before measurement, erythrocyte of chicken was used as standard sample to regulate coefficient of variation (CV) less than 5%. DNA index (DI) was used to represent cellular DNA content, which is the ratio of G0/G1 peak values in tumor cells and normal cells. It was considered a diploid tumor when the DI ranged from 0.85 to 1.15, or else it was a heteroploid tumor. Two fresh samples resected surgically were used to isolate tumor cells by using the technique of primary tissue culture. The cells were cultured in RPMI 1640 medium with 10% fetal cow serum (FCS) in the conditions of 37℃and 5% CO2. The cells were passaged 2 to 3 times to acquire purified tumor cells, which were then identified by Giemsa staining and immunohistochemistry against monoantibodies CK8 and smooth muscle actin. These two fresh samples were also detected by flocytometry. The purified tumor cells were seeded in 96-wells plate with 4×103 cells in each well, divided into control group and treatment group, and each groupincluded 4 wells. After adherence to the plate, the cells were transduced with HSV-tk (MOI=100), and treated with GCV (10-5mol/L) 24h later. After transfection for 3 to 5 days, the culture medium were discarded and 20 μL MTT (5mg/ml) were added into each well to incubate for 4h at 37℃. Then MTT was discarded and 200μl DMSO was added to each well and incubated for 15min at room temperature. The OD values were measured by enzyme labeling instrument under 490nm wavelength to calculate the survival rate of tumor cells. 3 The effects of HSV-tk suicide gene and IL-2 gene on salivary pleomorphic adenoma cells 3.1 Recombinant adenovirus Two kinds of recombinant adenovirus vectors carried HSV-tk and IL-2 gene, pUC19-HSVtk and pCA13-hIL2 plasmids were constructed by Shanghai Xinji Biotechnologic Limited Company and identified with restriction endonuclease EcoR I, BamH I and EcoRI, SalI, respectively. After recombination and package, the titer of adenovirus was determined to be 3×1012pfu/L. Enhanced green fluorescent protein (EGFP) recombinant adenovirus, whose titer was 1×1012pfu/L, was used as control. 3.2 Culture of human salivary pleomorphic adenoma cells Freshly isolated human pleomorphic adenoma cells were cultured at 37°C with 5% CO2 in RPMI culture media, containing 10% fetal calf serum, product of Hyclone. Once the cells grew confluent, they were subdivided. Cells of 2 to 3 passages, purified tumor cells, were collected and evaluated by the immunohistochemical staining. 3.3 Determination of the transfection efficiency of adenovirus Tumor cells, with its density of 36×104/ ml, were inocubated into 6 well plate and transduced by EGRP recombinant adenovirus, with MOI at 50, 100, 200, respectively. After incubated at 37℃for 48h, green fluorescence labeling cells were regarded as positive, and the transfection efficiency of adenovirus was determined by utilizing the cytometry method. 3.4 Detection of gene expression by RT-PCRPrimer design, TK: P1: 5'-ATG GCT TCG TAC CCC TGC-3′; P2: 5'-TCA GTT AGC CTC CCC CTA C-3′. IL-2: P1: 5'-CCC AAG CAA TGG ATG ATT TGA TGC-3′; P2: 5'-TTT ATG GCG GGA GGT AGA CTG ACC-3′. Pleomorphic adenoma cells were transduced by HSV-tk gene with MOI at 100 and by IL-2 gene with MOI at the same level, respectively, 72h later, and the transduced cells were collected. Total RNA of transduced cells was extracted by SK1351 Test Kit, and reverse transcription and PCR (RT-PCR) was conducted by RT-PCR test Kit according to the protocol. 3.5 The therapeutic effects of HSV-tk/GCV system and IL-2 gene on pleomorphic adenoma cells Pleomorphic adenoma cells, 4×103cells per well, were inocubated into 96 well plate. Then the cells were divided into 4 groups: (1) control group (EGFP); (2) HSV-tk/GCV group; (3) IL-2 group; (4) co-transfection group (HSV-tk and IL-2 group), and each group included 4 wells. Five days later, the survival rate of tumor cells was examined by utilizing the MTT method. 3.6 The by-stander effects of HSV-tk/GCV system and IL-2 gene on pleomorphic adenoma cells Tumor cells were transduced by different kinds of virus, all with MOI at 100, for 24h. Then infected cells and non-infected cells were mixed in various ratios and cultured in 96 well plate at a density of 4×103 cells per well. After 24h, GCV (10-5mol/L) was added to the mixtures. After 5 days culture, the survival rate of tumor cells was analyzed by MTT and the inhibition of cell proliferation was observed by microscope. Results 1 Primary cell culture of salivary pleomorphic adenoma and determination (1) The growth character and growth curve: SPA cells populated gently with longer adaptive phase. After 4-5 days cultured, the cells cleavaged and proliferated in large amount without apparently fast populated phase.(2) The character of morphology in primary cultured cells: There were many fragments of cells after tissue bit inocubated for 3-4 days without cell emigration. Until 7 days, cells expended gradually and formed a small area surrounding the primary tumor tissue under inverted microscope. The cells showed polygonal shape and flaky distribution whareas a minority of cells were dispersed. Besides some cells showed fusiform shape, a large amount of cells were polygonal. (3) The viability about transfer of cultured cells: Cells incubated for 17 days reached 80 % of cells confluenced, then subcultured firstly. After 48h the cells sticked wall as single dispersed ways or collective 3-5 number. Then, the cells cleavaged and proliferated to connecte finally. The morphology of the cells was fusiform and polygonal. (4) Observation of immunocytochemical staining: After Giemsa staining, neucleus showing purple red dye, cytoplasm showed pin dye. A glandular epithelium was round or columnar, arranged cluster and formed glandular structure. While, cytoplasm contained full mucus and constructed vacuole in particular cells. In addition, myoepithelial cells were fusiform or polygonal, distributed in single or lamellar. Some mucoid substance was arround the cells. (5) Observation of cytochemistry staining: After toluidine blue staining , the mucoid substance arround the cells showed purplish red dye with γmetachromasia, suggesting that the substances were connective tissue mucus. (6) Observation of immunocytochemical stain: A low molecular weight keratin antibody of anti-human was positive in columnar or round cells showing cytoplasm yellow-brown dye, suggesting that these cells were glandular epithelial cells. In addition, a smooth muscle actin antibody of anti-human was also positive in polygonal or fusiform cells showing cytoplasm brown-yellow dye, suggesting that the cells were amyoepithelial cells, distributed around the outside of glandular epithelial cells or in the mucoid substances. The glandular epithelial cells arranged lamellarly, theglandular lumen could be seen in some region. 2 The relationship between the gene therapy and ploid distribution in salivary pleomorphic adenomas 2.1 Results of flowcytometry analysis All of the 10 cases of normal salivary gland tissues were diploid. Among the 50 cases of pleomorphic adenoma, 40 cases were diploid, and another 10 cases were heteroploid. The frequency of heteroploid was 20%. One out of the 2 fresh samples was diploid and another 1 was heteroploid. 2.2 The relationship between ploid distribution and clinics Among the 50 cases of pleomorphic adenoma, the frequency of heteroploid was 33.3% among the patients less than 30 years old and 15.8% among the patients larger than 30 years old. The difference between these two groups was statically significant (P<0.05). The frequency of heteroploid was 21.9%, 16.7%, and 16.7% respectively when the tumor was located in parotid gland, submandibular gland, palate and other part. No statistical significance was observed among these three groups. There were 35 patients whose disease course was less than 2 years, and the frequency of heteroploid of them was 25.7%. The disease course of another 15 patients were more than 2 years and the frequency of heteroploid was 6.7%, which was markedly reduced as compared with the patients with shorter disease course (P<0.05). The frequency of heteroploid was 66.7% and 9.8% respectively between the tumor diameter more than 5cm and less than 5cm. The frequency of heteroploid was significantly enhanced as the tumor diameter increasing (P<0.05). 2.3 The effect of HSV-tk/GCV on pleomorphic adenoma After transfection of HSV-tk/GCV gene into pleomorphic adenoma cells of diploid for 3 days and 5 days, the survival rate of cells reached 78.3% and 37.7%. However, in the pleomorphic adenoma cells of heteroploid, the cellular survival rate was respectively 26.7% and 27.2% after transfection for 3 days and 5 days, which was obviously lower thanthat of diploid tumor (P<0.05). 3 The effects of HSV-tk suicide gene and IL-2 gene on salivary pleomorphic adenoma cells 3.1 Optimization of transfection conditions for adenovirus The transfection efficiency was defined as the ratio of cells containing the EGFP gene and non-containing the EGFP gene by cytomety. When MOI was 200,100 or 50, the transfection efficiency of adenovirus was calculated to be 51.4%, 49.1% and 26.7%, respectively. So, when MOI was 100, the highest transfection efficiency was obtained. 3.2 Detection of HSV-tk gene and IL-2 expression by RT-PCR After HSV-tk and IL-2 were transduced the pleomorphic adenoma cells, two PCR products of DNA fragments of HSV-tk and IL-2, 1150bp and 474bp could be seen by RT-PCR. But nothing could be observed in non-transduced cells. 3.3 The antineoplastic and by-stander effects of HSV-tk/GCV system and IL-2 on pleomorphic adenoma cells Five days later, the survival rate of cells transduced by IL-2 or HSV-tk/GCV was 66.0% or 37.7%, respectively. But the survival rate of cells transduced by both IL-2 and HSV-tk/GCV turned to be 21.5%, there was significant difference between IL-2 or HSV-tk/GCV transduced group and co-transduced group, (χ2 test, P<0.05). The by-stander effect in co-transduced group was higher than that in IL-2 or HSV-tk/GCV group, and when transfection efficiency was 75.0% and 50.0%, there was a significant difference between co-transduced group and IL-2 or HSV-tk/GCV gene transduced group (P<0.05). By microscope, the cells transduced IL-2 and HSV-tk/GCV, became to be shrunk; vacuole in the cytoplasm was observed, a part of them fell off the plate. But, there wasn't any obvious change in the control. Conclusion 1 The limited cell line of SPA construction (1) As compared with the primary tumour, monolayer cultured cells invitro may not form a double-layer duct constructure like the primary tumor tissues. This difference was related to the tumorigenesis. (2) In this experiment, the cultured cells may be similar to the primary tumor cells and showed myoepithelial and adenoepithelial differentiated. The cultured cells may not survive for a long time. This was not apparently fast growth period. (3) The cultured condition may be an important factor for epithelial cells to survive. The culture medium of RPMI1640 was better to foster the development of SPA cells than F12 medium. The effect of pro-growth factor on fostering cells development was very good. The subcultured cells of SPA maintain the characters of primary tumor cells. The histological feature was similar to the primary tumor. 2 (1) The frequency of heteroploid in the patients with pleomorphic adenoma was closely related with age, disease course, and tumor size. The frequency of heteroploid in the patients whose age was less than 30 years old and disease course was less than 2 years and tumor diameter was larger than 5cm, was 33.3%, 25.7% and 66.7%, respectively which was obviously higher than that of control group. These data demonstrated that the proliferation of the tumor cells in these patients was very active, and clinical doctors should pay more attention to them. (2) The pleomorphic adenoma cells of heteroploid were more sensitive to HSV-tk/GCV that could result in significant kill effect on these tumor cells. HSV-tk system might be an effective and patent measure for the treatment of salivary pleomorphic adenoma, especially for heteroploid ones. 3 (1) The anti-neoplastic effect of HSV-tk/GCV system was obvious in vitro. However, when IL-2 combined with HSV-tk/GCV, the killing effects were significantly stronger. (2) The by-stander effects were detected by using transduced single HSV-tk/-GCV or IL-2, but it was weaker in only IL-2. Not only the enhanced directly inhibitive and killing effects but also... |
