| This paper is composed of following three parts.PART ONE Biological behavior characteristics and their association with p53 signaling pathway in nasopharyngeal epithelia of TgN(p53mt-LMP1)/HT transgenic miceObjective:To investigate the biological characteristics and their association with p53 signaling pathway in nasopharyngeal epithelia of TgN (p53mt-LMP1)/ HT transgenic mice to aim at the exploration on their implications in the natural developing process of precancerous lesion in the nasopharynx. Methods: Included in this part of study were 36 animals randomly chosen from the generation of G10 TgN(p53mt-LMP1)/HT transgenic mice at the month age of 12th,15th and 18th respectively with positively expressed p53 and LMP1 activities, taken as 3 groups with 12 mice in each group. Another 3 groups of wild mice in the same strain were taken as the negative control group, with the same age animals in each group and at the same month age as well respectively. Then, their nasopharyngeal mucosa was taken to prepare samples for pathohistological determination in the consideration of biological behavior observation on the nasopharyngeal epithelia and immunohistochemical measurements on the expressive activities of cell cycle proteins with Western-blotting assays to confirm the conclusions got from immunohistochemistry respectively. Results: The tendency of precancerous lesions in the epithelia of nasopharyngeal and/or nasal mucosa of TgN(p53mt-LMP1)/HT transgenic mice was serious with the increase of the age and the shape of nuclear and mitochondrial was abnormal by the electro microscopy.The expression level of CDK2、cyclinD1 was increased significantly compared with the wild groups (P<0.01). There was no the expression of the p53 in the wild groups (western-blot).The results of the transgenic mice groups showed that there was an increasing tendency in the expressive levels of p53 protein in the mucosal tissue samples of nasopharyngeal and nasal epithelia with growing of mice at month age, as the animals at the age of 15th and 18th showing significantly higher protein level than that at the age of 12th month (P<0.05). This kind of tendency was also true in the expression of CDK2 and cyclinD1proteins, with significantly higher expressive levels among the transgenics mice at the age of 18th month than that of mice at 12th month (P<0.05). In the contrast, there was a decreasing tendency seen in the expressive activity of p21 protein in these tissue samples while there were no markedly statistical significance among different month-age groups (P<0.05). The morphological changes in the epithelia of nasopharyngeal and/or nasal mucosa were in the same developing tendency as well among different groups at various growing age of month. The correlation anlysis of P53、P21、CDK2 and cyclinD1、of the groups of the transgenics mice:P21-CDK2, P21-cyclinD1were negative correlation (P<0.05) and the CDK2-cyclinD1 was positive correlation (P<0.05).Conclusions:It can be concluded that the developing process of anabatic precancerous lesion in nasopharyngeal and/or nasal epithelia was possibly associated with the decreased expressive activity of P21 and increased activities of p53, CDK2 and cyclinD1 in these epithelia among the animals of TgN(p53mt-LMP1)/HT transgenic mice.PART TWO The intervening effect of Qi-boosting toxin-resolving formula on the precancerous lesion-inducing process of DNP among TgN (p53mt-LMP1)/ HT transgenic miceObjective:To investigate the effect of Qi-boosting toxin-resolving formula (QBTRF) on the precancerous lesion-inducing process of carcinogenic agent N,N’-dinitrosopiperazine (DNP) among transgenic mice TgN(p53mt-LMP1)/HT but without the use of carcinogenic promoting agent 12-O-tetradecanoylphorbol (TPA). Methods:Seventy two transgenic mice of TgN(p53mt-LMP1)/HT were randomly divided into 3 groups, i.e. modeling group (MG), treating group (QG) with QBTRF and positive controlling group (TG) with tretinoin, with 24 animals in each group. The animals in MG were treated by 0.2 ml of normal saline in the way of intragastric administration, those in TG were treated by 0.2 ml of concentrated decoction of QBTRF by lavage, and ones in PCG treated by 0.062 mg of tretinoin in the same volume of solution for each mouse, all being treated once each day. For all these animals, DNP was given in a dose of 1.0mg/10 g each time by subcutaneous injection, two times per week and lasted for 14 weeks, with a total injection number of 28 times. Moreover,24 wild mice in the same strain were taken as the normal controlling group (NG), without any additional intervening treatment for them other than the use of carcinogenesis-inducing agent in the same way as above. Then, these mice were put into death to collect tissue samples from their nasopharyngnx and nasal cavity for pathohistological observation of precancerous lesion and expressive activity determination of p53, P21, CDK2 and cyclinD1 respectively. Results: The epithelia of nasopharyngeal and/or nasal of MG had the charactstics of malignant by pathohistological and the change of plasma membrane had the structure of pseudopodium, and the shape and structrue of mitochondrial was abnormal by the electro microscopy. QG% TG and NG had no the changes.The expressive level of protein P21 was significantly higher in QG than that in NG with statistical significance (P<0.05), and markedly decreased expressive level of protein cyclinD1 in QG than that in MG, also with statistical significance (P<0.05). Meanwhile, the expressive levels of proteins p53 and CDK2 were significantly declined in QG when compared with that of other groups (P<0.05). All these indexes were companied by a paralleled trend of changes in the pathohistology in the epithelia of nasopharyngeal and/or nasal mucosa among different groups. Conclusions:It may be true that the intervening effect of herbal medicine QBTRF on the development of precancerous lesion in nasopharynx and/or nasal cavity of transgenic mice TgN (p53mt-LMP1)/HT should be brought about via the main pathway of down-regulating the expression of cyclinDl as well as p53, companied with up-regulating expression of P21.PART THREE Analysis on the development of tumors grown in the parts outside the nasopharynx and/or nasal cavity among TgN(p53mt-LMP1)/HT transgenic mice received the inducing treatment of carcinogenic agent DNPObjective:To analysis the possible mechanisms underlying the development of tumors grown in the other parts outside the nasopharynx and/or nasal cavity among TgN(p53mt-LMP1)/HT transgenic mice received the treatment of carcinogenic agent DNP in order to induce precancerous lesion in the nasopharynx. Methods:An overall review was made to the animals grown some tumors in the other parts outside the nasopharynx and/or nasal cavity among the mice as studied in Part Two for investigation on the intervening effect of herbal medicine QBTRF on the inducing process of DNP among them, mainly the morphological observation among them. Results:These tumors were most frequently seen among the animals in MG (8 mice with hapatoma,6 with fibrosarcoma,4 with lung cancer, and 9 with other kind of tumors, with an incidence of 62.50%), while only one with hapatoma and 4 with fibrosarcoma, with an incidence of 29.16% in QG. Moreover, there were 1 with hapatoma and 5 with fibrosarcoma in TG, with an incidence of 29.16%. However, there were 3 with fibrosarcoma and 4 with other kinds of tumor in NG, with an incidence of 20.80%. The most frequently seen lesions were hypatoma and fibrosarcoma, with a few animals with such a lesion in spleen diagnosed as mtastatic hypatoma but without primary liver carcinoma found. Moreover, one mouse was found with the neoplasm of mature teratoma in genital system, while a large part of lesion here was diagnosed as cystis degeneration in genital gland. Conclusions: This kind of tumor outside of nasopharynx and/or nasal cavity among TgN (p53mt-LMP1)/HT transgenic mice may be induced by the carcinohenesis-inducing effect of carcinogenic agent on the background of constitutional factor due to transferred genes p53mt and LMP1 through the inheritant way of vertical transmission and leading to a much sensitive situation in the whole body to the carcinogenic agent. |
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