Wood On Pg Cell Proliferation, Adhesion, And Research On The Effects Of Invasive Ability

Metastasis is the substantive characteristics of biological behaviour in malignant tumor, it is also the important standards in both the classification of clinical tumor and the judgment of malignant grade. So to research the metastasis of tumor is one of the most important tasks in the tumor prevention and treatment. The TCM theory sums the treatment principle up to4points according to the mechanism of malignant tumor: strengthen the healthy qi to prop up the root, detoxify and eliminate the stasis, clear the heat and dispel the dampness, reduce the phlegm and resolve masses. The method of promoting blood circulation to remove blood stasis has been widely researched in recent years. Many basic experiment and clinical researches have proved that most patients with tumor have the syndrome of "blood stasis". Thus has been proved throμgh the patients’clinical manifestation, abnormality of hemorheology, microcirculation disturbance and the hypercoagulable state of blood. The syndrome of blood stasis is not only the objective pathological state of the patients with malignant tumor, but also it may be the favorable factor of the cancer cells metastasis, it goes throμghout different pathological phases of the occurance, proliferation, infiltration and metastasis of the tumor. The higher malignant level of the tumor and later phases are, the more obvious high-condensation phenomenon is. So the herbs for invigorating blood circulation and eliminating stasis are widely used in tumor treating process. But some basic experiments and clinical researches have shown a very different outcome, the provement indicates that herbs for invigorating blood circulation and eliminating stasis may promote the metastasis of the tumor. And some part of the researches have shown that some kind of herbs for invigorating blood circulation and eliminating stasis can inhibit the proliferation and metastasis of the tumor. Then whether the herbs for invigorating blood circulation and eliminating stasis is a promoting factor or an inhibiting factor for the tumor may have a relationship with the different combination with other methods, like qi fortifying method, promoting the circulation of qi method and so on. it may also be related with the modern medical intervene such as operation, chemoradiotherapy, and the different applicative order of the herbs, and it also related with the own function of the herb and the method of the experiment. So far, the correct conclusion is difficult to get in all, so the further research must be continued to study the herbs for invigorating blood circulation and eliminating stasis to treat the tumor. This subject is based on the early stage of the tutor for further research with the help of NSFC. The observation was done in vivo test by watching the medical effect of Radix Salviae Miltiorrhizae, Caulis Spatholobi, sanguisμge, Ligusticum chuanxiong, Sappanwood towards the metastasis of the lung cancer in the early stage. the result is shown that the Sappanwood has an obvious inhibitive function to the metastasis of tumor. So we choose the total extracting solution of Sappanwood to intervene the PG cells and inoculate it into the nude mice to make the spontaneous metastasis occur. Throμgh testing the weight of the tumor in situ, the tumor-inhibition rate, the expression of E-cad of tumor in situ, the metastasis number of lung, the rate of lung metastasis, the expression of CD44v6of lung, P-select of serum, VEGF of liver and so on, the experiments in vitro takes MTT method, FCM testing CD29, reconstructing the basement membrane invasion and so on. It explains the effect of Sappanwood towards PG cells’proliferation, invasion and metastasis ability from different points of view, and then analyze the mechanism of tumor inhibited by Sappanwood, and it provides the scientific evidence that whether the herbs for invigorating blood circulation and eliminating stasis can be used in clinical affairs when treating a malignant tumor patient.Research methodExperiment in vivo:With different concentrations of sappanwood overall take liquid in the often oxygen (5%CO2,5%air) and low oxygen (5%O2,5%CO2,5%N2) environment intervenes PG cells, meanwhile set up blank control group, DDP group.Each cell inoculation in nude mice after drug intervention right axillary,6weeks to be put to death animals, lung, liver, and tumor with immunohistochemical methods were used respectively to detect the expression of CD44v6and VEGF, E-cad.Experiment in vitro:Establishing constant oxygen and low oxygen giant cell lung cancer cell growth, adhesion and invasion model, with different concentrations of sappanwood overall take liquid to intervene, meanwhile set up blank control and cisplatin. Tested by the MTT method its influence on PG cell proliferation capacity, flow cytometry to detect the effect of PG cells expressed CD29, infiltrating small room (transwell) model was used to study its effects on PG cell adhesion and invasion.Results1. Determined by MTT detect proliferation:when often oxygen24h, all of the experimental group compared with negative groups and cisplatin group were no significant differences (P>0.05); when Low oxygen24h cisplatin group difference compared with negative normal group (P<0.05), the experimental group compared with negative control group has a different degree of inhibition, but without statistical significance (P>0.05); when48h often oxygen,all of the experimental group compared with negative groups have obvious inhibitory effect (P<0.05), cisplatin and each experimental group also has a difference (P<0.05); Hypoxia at48h in addition to200and100μg/ml group, the rest of the experimental group compared with negative groups significantly inhibitory effect, there is statistical significance (P<0.05). Low oxygen and often oxygen compared with little influence on the proliferation of PG cells, and at48h sappanwood on proliferation of PG cells have obvious inhibitory effect.2. Flow cytometry detection expressed CD29:oxygen groups often expressed CD29average fluorescence intensity under low oxygen group, but no statistical significance (P>0.05). In normal oxygen and low oxygen environment, each experimental group and control group, each group between PG cells CD29average fluorescence intensity had difference (P<0.05), the middle concentration of the extract of sappanwood and cisplatin is no differences on PG cell expressed CD29(P>0.05).3. Tumor in situ tumor weight and tumor inhibition rate:In normal oxygen environment sappanwood group in tumor significantly lower than the control group, with statistical difference (P<0.001); In normal oxygen environment, sappanwood group compared with DDP group also has a difference (P<0.001), of which the tumor inhibition rate of sappanwood group was21.20%, tumor inhibition rate of DDP group was83.80%, sappanwood group than DDP group. Sappanwood group and the control group, DDP group in low oxygen also have compared difference (P<0.001), with tumor inhibition rate of sappanwood57.30%, tumor inhibition rate of DDP group90.30%. Compared to normal oxygen and low oxygen environment, between sappanwood group, DDP group had no difference (P>0.05), the difference between the control group (P<0.001).4. CD44v6expression in lung tissue:Sappanwood group and cisplatin group expression of CD44v6is lower than the normal oxygen environment in low oxygen environment, there was an obvious difference, all had statistical significance (P<0.05). Sappanwood group expressed in normal oxygen environment is often higher than in cisplatin group, but no statistical significance (P>0.05). The Sappanwood group in low oxygen environment expression was lower than cisplatin group, with statistical significance (P<0.001), Sappanwood and cisplatin group in two environment has the obvious difference with control group (P<0.001). That Sappanwood and cisplatin group in terms of CD44v6expression effect has obvious inhibitory effect, the low oxygen environment Sappanwood than cisplatin group stronger inhibitory effect.5. expression of VEGF in liver tissue:negative control group expression of VEGF in low oxygen environment was significantly higher than often oxygen environment, and there is statistical significance (P<0.05); Sappanwood group expression of VEGF of normal oxygen is significantly higher than the low oxygen environment, with statistical difference (P<0.05); Cisplatin group expression of VEGF in low oxygen environment is higher than the normal oxygen environment, but no statistical difference (P>0.05). In often oxygen environment Sappanwood group and cisplatin group were higher than control group, both have difference (P<0.001), and in low oxygen environment Sappanwood group had no obvious difference (P>0.05). That Sappanwood and cisplatin group under normal oxygen environment has obvious promoting angiogenesis effect, and in low oxygen environment,Sappanwood has no obvious effect.6. expression of E-cad in situ tumor:Sappanwood group, cisplatin group in E-cad expression on oxygen environment were higher than the negative control, low oxygen environment lower than the negative control group, but under the environment of two kinds of no statistical significance (P>0.05). Illustrate sappanwood on the expression of E-cad and cisplatin group were no obvious influence.7. Invasion experiment:low concentration group and normal control group in the low oxygen environment in accumulated optical density value is higher than the normal oxygen environment, normal concentration, high concentration group and in the cisplatin group under normal oxygen environment in accumulated optical density value is higher than the low oxygen environment, but the two oxygen environment is no significant difference (P>0.05). In normal and low oxygen environment, high, medium and low concentration of sappanwood group and the cisplatin group in accumulated optical density value were significantly lower, downward trend was dose dependent, which the cisplatin group in accumulated optical density value is lower than the concentration in the group, but no statistical significance (P>0.05). Each test group in accumulated optical density value compared with normal control group were statistically significant, in the accumulated optical density value in addition to the middle concentration and cisplatin group no difference, the rest all have statistical significance (P<0.001).Comprehensive the above results, we think the sappanwood have significant inhibition of tumor, including inhibition of tumor proliferation, adhesion and invasion process. Including proliferation phase in two kinds of oxygen environment sappanwood has obvious tumor suppression effect, the low oxygen environment tumor inhibition rate higher; Adhesion phase is mainly to reduce cancer cell surface expression of integrin, namely reduce the adhesion of cancer cells and basement membrane process, and rely on the adhesion of calcium element expression, namely adhesion between cells had no obvious effect; Invasive stage found increased as the concentration of sappanwood tumor inhibition also increased obviously, into a dose dependence. In angiogenesis sappanwood can promote blood vessel formation, which is often especially in oxygen environment.