Dissertation
Dissertation > Medicine, health > Oncology > Respiratory system tumors > Lung tumors

Study on the mechanism of effect of the serum containing hematoxylin, Caulis Spatholobi on proliferation of PG cells

Author GuoXiuWei
Tutor ZhangPeiTong
School Beijing University of Traditional Chinese Medicine
Course Traditional Chinese Medicine
Keywords Lignum Sap pan Spatholobi cisplatin medicated serum cell cycle P16 Rb1
CLC R734.2
Type Master's thesis
Year 2014
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Traditional Chinese medicine held that blood stasis and Cancerous toxin intertwined and then produced cancer. Jingyue Quanshu says:"blood stasis detention produced mass", which explained that blood stasis syndrome was closely related to tumor formation. Modern medicine found that95%of tumor patients had one or more abnormality of clotting condition in laboratory examination through the investigation on high clotting and viscosity condition of the blood in cancer patients. Accordingly, stasis-dissolving drugs had been widely used in tumor treatment.Lignum Sappan and Spatholobi, as one of stasis-dissolving drugs, have been widely used in various tumor treatment including lung cancer in clinic. In recent years, research in vitro and in vivo indicated Lignum Sappan and Spatholobi had a certain inhibitory effect on various tumors. Researches on this topic in the past vivo experiments focused on effects on the growth and metastasis of different stages of lewis lung cancer in four kinds of stasis-dissolving drugs including chuanxiong, and Spatholobi, Lignum Sappan and leech and then the result also suggested that Lignum Sappan and Spatholobi had significant effect on suppressing tumor growth and metastasis. In the early vitro experiment, MTT assay was adopted to check effect of Sappan extract on PG cell proliferation in normoxic and hypoxic environment. The results found Sappan extract showed some inhibitory effect on the proliferation of PG cells in normoxic and hypoxic conditions, but that was less obvious in hypoxia environment than in normoxic condition. Experiments made in Beijing Chinese Medicine Hospital affiliated to Capital Medical University also found that spatholobi extracts have antiproliferative effect on six kind of tumor cell lines (A549, HT229, PG, PANC21, SMMC27721, IEC26)in vitro.Lung cancer was one of the common malignant tumor with poor prognosis and most patients had lost the best opportunity of surgical operation and multidisciplinary treatment. For decades, chemotherapy has been the basic measure of tumor therapy. Although chemotherapy can kill tumor cells, it can also kill non-tumor cells in the cell cycle such as intestinal epithelial cells, hair follicle cells and hemopoietic stem cell. These side effects could cause serious consequences which limited the application of chemotherapy. Therefore, one of the problems to be solved was to seek a new antitumor drug and thus to improve the quality of life and prolong the survival time in patients with lung cancer. This study adopted Lignum Sappan medicated serum, Lignum Sappan medicated serum plus cisplatin, Spatholobi medicated serum, Spatholobi medicated serum plus cisplatin, blank serum, cisplatin on human lung cancer PG cells cultured in vitro to observe the effect on the proliferation of PG cells, cell morphology, the percentage of each cell cycle, the expression of cyclinDl, CDK4and PCNA proteins and the expression of Rb, P16mRNA related with cell cycle and thus to provide the experimental basis for the anticancer mechanism of Lignum Sappan and Spatholobi.Objective:To explore the effect of Lignum Sappan and Spatholobi medicated serum plus cisplatin on growth inhibition of human lung cancer cell line PG, its proliferation cycle arrest and molecular mechanism.Methods:1Prepare Lignum Sappan and Spatholobi medicated serum and blank serum, and then MTT was adopted to observe the inhibitory effect of Lignum Sappan and Spatholobi medicated serum plus cisplatin on the proliferation of PG cells;2The light microscopy and laser scanning confocal microscope were used to observe the morphology of PG cells in blank group, Lignum Sappan group, Lignum Sappan plus cisplatin group, Spatholobi group, Spatholobi plus cisplatin group and cisplatin group respectively;3Flow cytometry was applied to observe the cell cycle arrest;4Laser scanning confocal microscopy was used to observe the effect of each group serum on the expression of PCNA, CyclinDl, CDK4protein in PG cells;5The expression of P16, Rbl mRNA was tested by PCR method.Results:1Each medicated serum group related with Lignum Sappan all appeared inhibition after24h drug intervention and had a dose and time dependence. Compared with Lignum Sappan group, Lignum Sappan plus cisplatin group and cisplatin group had strong inhibition. Lignum Sappan plus cisplatin group was obviously inhibited in comparison with cisplatin group, but had no statistical significance.2By the light microscopy and laser scanning confocal microscope, the apoptosis degree of PG cells in Lignum Sappan group was significant but less than Lignum Sappan plus cisplatin group as well as cisplatin group. Lignum Sappan plus cisplatin group was obviously inhibited in comparison with cisplatin group, but had no statistical significance.3Compared with blank group, G0/G1and S phases proportion of PG cells in Lignum Sappan group increased with G2/M phase decreasing; S and G2/M phases proportion in cisplatin group together with Lignum Sappan plus cisplatin group raised and G0/G1phase reduced, in which G2/M phase added and G0/G1phase decreased significantly compared with Lignum Sappan group. Each phase of PG cells in Lignum Sappan plus cisplatin group showed no significance compared with cisplatin group.4The expression of CyclinDl, CDK4, PCNA protein reduced in Lignum Sappan group compared with blank group. That in cisplatin group and Lignum Sappan plus cisplatin group decreased too and was lower than Lignum Sappan group; in comparision with cisplatin group, that in Lignum Sappan plus cisplatin group showed no difference.5The expression of P16, Rbl mRNA increased in lignum Sappan group compared with blank group. That in cisplatin group and Lignum Sappan plus cisplatin group raised too and was higher than Lignum Sappan group; in comparision with cisplatin group, that in Lignum Sappan plus cisplatin group showed no difference.6Each medicated serum group related with Spatholobi all appeared inhibition after24h drug intervention and had a dose and time dependence. Compared with Spatholobi group, Spatholobi plus cisplatin group and cisplatin group had strong inhibition. Spatholobi plus cisplatin group was obviously inhibited in comparison with cisplatin group, but had no statistical significance.7By the light microscopy and laser scanning confocal microscope, the apoptosis degree of PG cells in Spatholobi group was significant but less than Spatholobi plus cisplatin group as well as cisplatin group. Spatholobi plus cisplatin group was obviously inhibited in comparison with cisplatin group, but had no statistical significance.8Compared with blank group, G2/M phases proportion of PG cells in Spatholobi group increased with G0/G1and S phase decreasing, but had no statistical significance; S and G2/M phases proportion in cisplatin group together with Spatholobi plus cisplatin group raised and G0/G1phase reduced, in which G2/M and S phases added and G0/G1phase decreased significantly compared with Spatholobi group. Each phase of PG cells in Spatholobi plus cisplatin group showed no significance compared with cisplatin group.9The expression of CyclinDl, CDK4protein were the same in Spatholobi group and PCNA protein was reduced compared with blank group. That in cisplatin group and Spatholobi plus cisplatin group decreased and were lower than Spatholobi and blank group; in comparision with cisplatin group, that in Spatholobi plus cisplatin group showed no difference.10The expression of P16, Rbl mRNA showed no difference in Spatholobi group compared with blank group. That in cisplatin group and Spatholobi plus cisplatin group raised compared with blank group Spatholobi group; in comparision with cisplatin group, that in Spatholobi plus cisplatin group showed no difference.Conclusion:Lignum Sappan medicated serum had obvious inhibitory effects on tumor cells prol iferation which induced PG cell apoptosis by down-regulating CyclinDl, CDK4and PCNA protein expression, up-regulating P16, Rbl mRNA transcription then causing G0/G1、S phases arrest while cisplatin by regulating cyclinBl mRNA transcription then resulting G2/M、S phases arrest. Cisplatin can also produce obvious inhibitory effects on tumor cells proliferation by down-regulating CyclinDl, CDK4, PCNA protein expression, up-regulating P16,Rbl mRNA transcription then causing G2/M、S phases arrest.Spatholobi medicated serum had obvious inhibitory effects on tumor cells proliferation which cannot induce PG cell apoptosis by down-regulating CyclinDl, CDK4protein expression, up-regulating P16, Rbl mRNA transcription then causing GO/G1、S phases arrest, but by down-regulating PCNA protein expression then causing G2/M phases arrest indirectly while cisplatin by regulating cyclinBl mRNA transcription then resulting G2/M、S phases arrest. Cisplatin can also produce obvious inhibitory effects on tumor cells proliferation by down-regulating CyclinD1, CDK4, PCNA protein expression, up-regulating P16, Rbl mRNA transcription then causing G2/M、S phases arrest.

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