| Objective: previous studies have found that the topic of shittim TCM phlegm drug has obvious effect of liver cancer, Gleditsia sinensis extract could significantly control the cancer TGF- beta/Smads signal system and PTEN/PI3K/AKT signaling pathway. We speculated that Gleditsia sinensis extract may by regulating the expression of micro RNAs related to regulate related signaling molecules, so as to play the role of resistance of liver cancer. On the basis of the previous studies, through theoretical and experimental research, we choose the related micro RNAs and target genes for validation. To further clarify the molecular mechanism of the drug treatment of liver cancer, which will provide a scientific basis for Chinese medicine development and clinical application of shittim.Methods: The first part of the oretical research 1. The prevention and treatment of liver cancer has a long history of traditional Chinese medicine, phlegm and blood stasis in liver cancer progression of alternating knot through disease, phlegm method is an important therapy for the treatment of liver cancer, also should always carry out the treatment of liver cancer.2. The value of TCM phlegm medicine of shittim, characteristics and the ancient and modern clinical application. And Gleditsia sinensis extractis the prophase research basis of liver function.3. As the research progress of molecular biology for liver cancer, liver cancer molecular targeted therapy is one of the most active field of clinical research nowadays, under the guidance of TCM theory, combined with modern molecular biology techniques, to clarify the mechanism of TCM prevention and treatment of liver cancer, is the development direction of traditional Chinese medicine for liver cancer prevention and control, also helps to find new targets for the treatment of liver cancer. Preliminary study based on role. The second part of the experimental research1. Walker- 256 portability rat model of liver cancer Will first Walker- 256 cancer cells cryopreserved cryopreserved tube out, quickly put it in the 37 water bath slot, after being its melt com℃ pletely, then remove freeze tube, and beat upon cell suspension in a sterile environment, transfer the cell suspension to 15 ml of centrifuge tube. Gently shake the centrifugal tube, slowly add PBS buffer to 8 ml. Then the centrifugal pipe centrifuge for 10 min at a speed of 1000 r/min, abandon supernatant, wash 2 times with PBS buffer. Then with 4 ml PBS buffer heavy suspension cells, with a 5 ml syringe injection respectively only Wistar rats abdominal cavity, each 1 ml. Department of 20 days to observe rat’s abdomen, cancerous ascites formation.From represented the Wistar rats extracting red meat wash water samples 15 ml, cancerous ascites in centrifuge centrifuge at a speed of 2000 RPM/min, 2 min after the completion of the upper left to pale clear liquid, and keep the lower viscous concentrated ascites, washed 3 times with PBS buffer, made into density of 2 x107 / ml cells suspension. Set aside.Will rise to about 65 only about 200 g SD rats by weight average divided into 6 groups, namely blank group, model group, high dose group of shittim extract, honeysuckle extract dose group, low dose group of shittim extract and sorafenib group, 10 in each group. Another five validation as the build mode and standby. Experimental rats fasting 12 hours before surgery, the model group, high dose group of shittim extract, honeysuckle extract dose group, low dose group of shittim extract and sorafenib group respectively according to 40 mu g / 100 g weight chloral hydrate anesthesia, the abdominal cavity is fixed in the supine position after rat board, 0.5% energy iodine disinfection abdomen, along the white line cut open the abdomen and abdominal separation step by step, exposed rats abdominal cavity, visible liver bulging belly. Select the outer lobe, 1 ml with a syringe cancerous ascites cell suspension, will be equipped with cancerous ascites syringe into the needle into the lobe is about 0.5 cm, gently push the syringe injection preparation of cancerous ascites is about 0.15 ml. After quickly pull out the syringe needle, with disinfection swab on the puncture point until no bleeding. After Hai Na liver into the abdominal cavity, step by step a closed abdominal cavity. The wound using erythromycin ointment to wipe disinfection.To prove building success, all 53 model rats after the completion of the building, only 7 days after building, in which random 3 only, another 12 normal rats choose 2, remove the liver of rats, to observe the pathological section.2. The treatment measures According to clinical dosage(acacia 1.5 g content. 70 kg. D- 1), give the dose based on reference [26], and convert the equivalent dose calculation of rats body surface area to the experimental dose lavage(conversion coefficients in the following table, the body standard of the average weight is 70 kg, the standard of the average weight is 200 g) of rats, according to the low dosegroup shows proximately 1:2:4 as the set of shittim extract(128.57 mg) kg) d-1), middle dose group(257.14 mg) kg) d- 1) and high dose group(514.28 mg) kg) d- 1); Sorafenib: 68.57 mg/kg/d(sola than adult 0.8 g/d, according to convert the equivalent dose calculation of human and mouse body surface area); Blank group was given 0.9% saline 10 ml/kg/d; Model group: to establish a model of liver cancer after dosing with the blank group; Groups of lavage dosage are the same size; 1 weeks after transplantation treatment; A laparoscope, usually connected to a group of 28 d. Termination of the treatment time for 35 d after transplantation.Line will be drawn in the rat carotid push dislocated executed under aseptic conditions, after disinfection were open, blank for rats structure integral part of the selected set of liver specimens; After building of rats, the selection in the liver cancer tissue, such as liver cancer nodules is, try to take the biggest part; Retained after tissue will be removed with filter paper blot, organization to be detected packet processing, cut into the range of 1 cm x 1 cm, 0.2 cm thick tissue block, serial section, ethanol mixture with formaldehyde fixed for 3 hours, and then fixed automatic dehydration, for HE staining and Tunel apoptosis detection rat liver pathological section.3. The experimental methods Experiment 1: the observation group rats tissue pathological morphology change; Groups observed by TUNEL apoptosis detection technology in the organization cell apoptosis, cell apoptosis rate calculation, statistical analysis. Experiment 2: use Real- time PCR detection technology to detect each group of rats in mi R- 21, mi R- 181- b, the expression of mi R- 183, carries on the statistical processing.Experiment 3: using Western blot assay and Real- time PCR assay, to detect each group of rats, PTEN, TIMP3, MMP2, MMP9, PDCD4 protein and m RNA expression, by statistical processing, comparison and analysis.Results: Experiment a Gleditsia sinensis extracton rat liver cancer pathology organization form1. From the perspective of morphological observation Group normal lobular architecture are clear and complete uniform cell cytoplasm nucleus, liver cell line, order to radiate out in central vein as the center. Nucleus is bigger, in central liver cells, the state uniform. Hepatic sinus and portal area within a small lymphocyte infiltration and hyperplasia of fibrous tissue.And lobular lost normal structure in model group, most of the hepatic lobule replaced by tumor tissue, peripheral lobular compression quality of a material change, hepatic sinus expansion, hepatic blood sinus surrounding fibrous tissue hyperplasia. Collect abbacy broadening obviously, there are a large number of fibroblasts, fiber cells. Tumor cells and rounded, regeneration active and pleomorphism, nuclei are darker and larger nuclear/cytoplasmic ratios, and part of a nuclear pyknosis and nuclear fragmentation phenomenon, are distributed in tumor cells crumb, some part of coagulation necrosis, dyeing dark red.Will Gleditsia sinensis extractlow dose group of pathological compared with model group, while hepatic lobules lost normal structure, part of the liver lobules was replaced by the tumor tissue; But cells form a little improvement, but still appear serious nuclear pyknosis phenomenon, a large nuclear/cytoplasmic ratios, and part of the cell is crumb. Hepatic sinus expansion, liver blood sinus surrounded by fibrous tissue hyperplasia.Gleditsia sinensis extractdose group compared with model group, a greater degree of improvement, can clearly see that the structure of hepatic lobule had certain recovery, tumor tissue, cell number and a plastic pleomorphic cells decreased, karyoplasm larger cells also decreased significantly. Hepatic blood sinus surrounding occasionally to fibrous tissue hyperplasia.Gleditsia sinensis extracthigh dose group, look from the morphological structure of hepatic lobule are clear and complete basic cell cytoplasm nucleus even, most of the normal hepatic lobule cells, some cells have the phenomenon of nuclear pyknosis and atypia, but compared with model group, the condition improved obviously. Rarely seen liver blood sinus surrounding fibrous tissue hyperplasia.Mr Fini group of morphology change is most obvious in the model group, and the most close to the normal group, hepatic lobule structure are clear and complete basic cell cytoplasm nucleus even, neat liver cells and to radiate out in central vein as the center. Larger nucleus, located in the liver cells in the central, the overall distribution more uniform. Hepatic sinus and portal area within a small lymphocytic infiltration, basic no fibrous tissue hyperplasia. Compared with the normal group, there are very few cells appear atypia, no clumps distribution phenomenon.2. Using TUNEL apoptosis detection technology, observation group 6 slices of cell apoptosis, each group took four horizons, microscopic biopsy on apoptosis cells to tan, image analysis system for quantitative analysis of liver cancer cell apoptosis index. Through one-way ANOVA test, P = 0.000, P < 0.01, n = 24, six more significant difference between groups, each group two comparison, Gleditsia sinensis extractgroups with sorafenib group comparison, P = 0.274, P > 0.05, there was no statistically significant difference. All have significant difference between groups(P = 0.000, P < 0.01).Experiment 2 Gleditsia sinensis extracton rat liver cancer of micrornas1. Through the Real- time PCR detection mi R- 21 the expression level of liver cancer cells. Through the single factor analysis of variance, the expression of 6 groups of mi R- 21 quantity has significant difference(P = 0.000, P < 0.01). Comparison between the two groups of two, in addition to Gleditsia sinensis extracthigh dose group and sorafenib group there was no statistically significant difference compared(P = 0.975, P > 0.975). All have significant difference between groups(P < 0.01).2. Through the Real- time PCR detection of mi R- 181- b in liver cancer cells, the expression level. Through the single factor analysis of variance, the expression of the six groups of mi R- 181 b is quantity is significant difference(P = 0.000, P < 0.01). Comparison between the two groups of two, in addition to Gleditsia sinensis extracthigh dose group and sorafenib group there was no statistically significant difference compared(P = 0.954, P > 0.954). All have significant difference between groups(P < 0.01).3. Through the Real- time PCR detection of mi R- 183 in liver cancer cells, the expression level(see table 4 and figure 7). Through the single factor analysis of variance, the expression of 6 groups of mi R- 183 amount have significant difference(P = 0.000, P < 0.01). Comparison between the two groups of two, in addition to Gleditsia sinensis extracthigh dose group and sorafenib group was statistically significant(P = 0.023, P < 0.05). All have significant difference between groups(P < 0.01).Experiment three Gleditsia sinensis extracton rat liver cancer PTEN and TIMP3, MMP2, MMP9, PDCD4 m RNA and protein1. Application QRT- PCR and Western blot test in rats of liver cancer in the expression of PTEN m RNA and protein levels in the organization, through the single factor analysis of variance, 6 groups of PTEN m RNA and protein expression level had significant difference(P = 0.000, P < 0.01). PTENm RNA between each two comparison, Gleditsia sinensis extracthigh dose group and sorafenib group there was no statistically significant difference compared(P = 0.365, P > 0.365). Groups of PTEN protein between two comparison, model group and honeysuckle extract low dose group(P = 0.011, P < 0.05), high dose group and Gleditsia sinensis extractsorafenib group there was no statistically significant difference compared(P = 0.076, P > 0.076), between groups had significant difference(P < 0.01).(2) in the detection of liver tissue of rats TIMP- 3 m RNA and protein expression level, through the single factor analysis of variance, 6 groups of TIMP- 3 m RNA and protein expression level had significant difference(P = 0.000, P < 0.01). Comparing two groups between TIMP- 3 m RNA, Gleditsia sinensis extracthigh dose group and sorafenib group there was no statistically significant difference compared(P = 0.064, P > 0.064). Comparing two groups between TIMP- 3 protein, Gleditsia sinensis extractdose group and low dose group of shittim extract comparison(P = 0.017, P < 0.05), high dose group and Gleditsia sinensis extractsorafenib group there was no statistically significant difference compared(P = 0.054, P > 0.054), between groups had significant difference(P < 0.01).(3) in the detection of liver tissue of rats MMP2 m RNA and protein expression level, through the single factor analysis of variance, 6 groups of MMP2 m RNA and protein expression level had significant difference(P = 0.000, P < 0.01). Comparing two groups between MMP2 m RNA, compared with model group and low dose group of shittim extract statistically significant(P = 0.015, P < 0.05), Gleditsia sinensis extracthigh dose group and sorafenib group there was no statistically significant difference compared(P = 0.996, P > 0.996). MMP2 proteins between two groups in terms of two comparison, compared with the blank group and sorafenib group statistical difference(P = 0.04, P < 0.05), model group and honeysuckle extract low dose group(P = 0.036, P < 0.05), high dose group and Gleditsia sinensis extractsorafenib group there was no statistically significant difference compared(P = 0.129, P > 0.129), the rest were significant differences between groups(P < 0.01).4. In the liver tissue of rats MMP9 m RNA and protein expression level, through the single factor analysis of variance, 6 groups of MMP9 m RNA and protein expression level had significant difference(P = 0.000, P < 0.01). Comparing two groups between MMP9 m RNA, compared with model group and low dose group of shittim extract statistically significant(P = 0.032, P < 0.05), high dose group and Gleditsia sinensis extractsorafenib group there was no statistically significant difference compared(P = 0.859, P > 0.859). MMP9 protein between each two comparison, compared with the blank group and sorafenib group statistical difference(P = 0.035, P < 0.05), model group and honeysuckle extract low dose group(P = 0.015, P < 0.05), low dose group and middle dose group(P = 0.013, P < 0.05) difference was statistically significant, Gleditsia sinensis extracthigh dose group and sorafenib group there was no statistically significant difference compared(P = 0.189, P > 0.189), between groups had significant difference(P < 0.01).5. Detection of cancer of the liver tissue of rats in PDCD4 m RNA and protein expression level, through the single factor analysis of variance, 6 groups of PDCD4 m RNA and protein expression level had significant difference(P = 0.000, P < 0.01). Between groups of PDCD4 m RNA pairwise comparison, compared with model group and low dose group of shittim extract statistically significant(P = 0.025, P < 0.05), both two comparison between groups had significant difference(P < 0.01). Between groups of PDCD4 protein both two comparison with significant difference(P < 0.01).Conclusion: 1. The Gleditsia sinensis extractcan induce Walker- 256 portability rat liver cancer cell apoptosis, has significant effect of liver cancer, of which the most obvious effect of high dose group, and no obvious difference was found between sorafenib.2. Gleditsia sinensis extract in the process of induced rat liver cancer cell apoptosis, can cut mi R- 21, cancer of the liver cells and increase the expression of tumor suppressor gene PTEN gene and protein; Still can make the mi R- 181 b, at the same time affect TIMP3(matrix metalloproteinases inhibitory factor 3)/MMP2(matrix metalloproteinases 2)/MMP9(matrix metalloproteinases 9) system; Can also be cut mi R- 183, at the same time, raised its target- suppressor PDCD4(4) programmed cell apoptosis factors, of which the most significant effect of high dose group, and sorafenib group was no significant difference.3. Through the acacia mentioned content can regulate the expression of micrornas and their corresponding target genes, further verify the Gleditsia sinensis extractpromotes liver cancer cell apoptosis mechanism, further verify the multiple targets of Gleditsia sinensis extractanti-cancer effect. |
PDF Full Text Request