| Objective:Lung cancer is a common malignant tumor,Although surgery radiotherapy and chemotherapy of comprehensive therapy for lung cancer has made great progress,However,a result of chemotherapy drugs multi-resistant phenomena and serious adverse reaction,lung cancer treatment failed to achieve satisfactory results,Targeted therapy of lung cancer has opened up a new way, but there are many problems,such as lung cancer group mutation rate is low,higher prices also limit the use of these drugs.Therefore,research on Chinese anti-tumor mechanism of action has important theoretical significance and application value.Xiaoaiping for medicine Marsdenia(Marsdenia tenacissima) extract,has been widely used in clinical,commonly used in lung and gastrointestinal tumors,The mechanism of its action so far, domestic scholars have conducted a series of research,Xiaoaiping and cell cycle-specific drugs less joint applied research.This experiment was to study the impact of Xiaoaiping lewis lung growth and cell cycle specificity combined use of anti-tumor effect,for Xiaoaiping applied to lung cancer treatment to provide some new theoretical basis.Methods:Preliminary experiments:(1)The exponential growth of 0.2 ml lewis unicellular suspension(cell concentration,106 / ml) subcutaneous grown in C57BC/L mice axillary,a total number of 10,only observe the survival of mice and the state of the time.(2) Copy Lewis lung cancer model in mice, again with the above method number, a total of 30,The above mice were randomly divided into two groups of 15,On the 10 th day intraperitoneal injection of physiological saline r e s p e c t i v e l y 0. 2 m l / o n l y, X i a o a i p i n g i n j e c t i o n( 0. 2 m l / o n l y, 4 0mg/ml),Respectively, in the first twelve days,fourteen days and sixteen days,mice were sacrificed 5,After the removal of part of the tumor tissue,cut into pieces,Adding a small amount 0.01 mmo L / l phosphate buffer,with 300 mesh nylon mesh filter,the filtrate centrifugal,abandon supernatant, PBS was washed three times,each tube with 1 ml75 % ethanol,and shocks after single fixed, at minus 20 degrees cryopreserved,determination of computer before recovery,centrifugal to remove ethanol, PBS rinse 2 times,each tube to join prepared with normal saline containing 0.5% Triton X- 100,1% of RNA enzymes,5% mixture of PI 0.5 ml, with 300 nylon mesh filter after 30 min, f i l t r a t e, t h e c e l l c y c l e b y f l o w c y t o m e t r y i n s t r u m e n t a n a l y s i s.Experiment:(1)experimental animal group and drug delivery:Copy the Lewis lung carcinoma mouse model, a total number of 60,the mice modeling were randomly divided into six groups of 10,On for 10 days after planting, according to one mouse for the dose to the dose conversion formula calculation,its dosage and times are as follows :One Group :Physiological saline only 0.2ml/( intraperitoneal injection of d10- d28);Two groups:Xiaoaiping only 0.2ml(Xiaoaiping injection 40mg/ml, intraperitoneal injection of d10- d28);Three groups: Fluorouracil only 0.2ml(Fluorouracil 3mg/ml d14 d18, d22, d25 intraperitoneal injection,the other time with Physiological saline, intraperitoneal injection, to 28 days);Four groups:Xiaoaiping only 0.2ml(Xiaoaiping injection 40mg/ml, intraperitoneal injection of d10-d14),14 days start with Fluorouracil, Fluorouracil only 0.2ml(Fluorouracil 3mg/ml d14,d18,d22, d25 intraperitoneal injection, the other time with Xiaoaiping, to 28 days);Five groups: Mitomycin only 0.2ml(Mitomycin 0.04mg/ml d14, d18, d22, d25 intraperitoneal injection,the other time with Physiological saline, intraperitoneal injection,to 28 days);Six groups:Xiaoaiping only 0.2ml(Xiaoaiping injection 40mg/ml, intraperitoneal injection of d10-d14),14 days start with Mitomycin, Mitomycin only 0.2 ml(Mitomycin 0.04 mg/ml d14, d18, d22, d25, intraperitoneal i n j e c t i o n, t h e r e s t o f t h e t i m e w i t h X i a o a i p i n g, t o 2 8 d a y s).(2)Observation group growth situation:every 3 to 4 days with vernier caliper measurement of tumor size(a) and vertical diameter(b),tumor size = ab2/2,mice tumor growth inhibition rate calculation,drawing tumors volume growth curve.(3)VEGF immunohistochemical:12h after tumor tissues with formalin fixed,paraffin embedding set aside,in strict accordance with the manual operation,the determination of vascular endothelial growth factor(VEGF) expression.Protein expression of quantitative results at high magnification(will) positive cells positive area percentage(%), randomly selected from each section 5 horizons,averaging.VEGF protein expression positive judgment: staining intensity: negative 0,light brown 1 minute,tan 2 points,deep tan 3 points;B positive cells: A third below for 1 minute, I / 3 ~ 2/3 of 2 points,more than two-thirds integral number for 3,A * B = 0 negative for A X B = l ~ 4 divided into weakly positive,A X B > 4 divided into strong positive,high expression.(4)Statistic analysis:all data using SPSS13.0 for Windows software is analyzed,to explore Xiaoaiping inhibitory and Inhibitory effect of Xiaoaiping combined with cell cycle-specific chemotherapy drugs.Results:1 A tumor of time:Copying Lewis lung cancer cells model,the mice 9 days after inoculation of tumor can hit millet grain size of nodules.2 Cell Cycle:The first twelve days Xiaoaiping group G1:0.3220 ± 0.02950, the saline group G1:0.3340 ± 0.0336, pairwise comparisons P>0.05, no significant difference,The first fourteen days Xiaoaiping group G1:0.5540 ± 0.04827,the saline group G1: 0.2900 ± 0.03536,pairwise comparisons P<0.05,The first sixteen days Xiaoaiping group G1: 0.5920 ± 0.03701,the saline group G1: 0.2980 ± 0.04324, pairwise comparisons P<0.05,twelve days Xiaoaiping group G1:0.3220 ± 0.02950 and fourteen days Xiaoaiping groupG1:0.5540 ± 0.04827,pairwise comparisons P<0.05,fourteen days Xiaoaiping group G1:0.5540 ± 0.04827 and first sixteen days Xiaoaiping group G1: 0.5920 ± 0.03701,pairwise comparisons P>0.05. Although G1 phase sixteen days is fourteen days G1 phase is high, but the comparative indifference.3 Tumor size:On 9 days after inoculation can hit millet grain size of tumor nodules,18 days before the tumor diameter was no significant change in 18 days visible tumor volume greater than the Saline group in each experimental group,in the Saline group tumors larger than 28 days in each experimental group,Xiaoaiping group is greater than the rest of the experimental group,Mitomycin group and Xiaoaiping combined with Mitomycin have no significant difference, Fluorouracil, Xiaoaiping combined with Fluorouracil and Xiaoaiping also have significant difference.4Tumor growth inhibitionrate and tumor:Saline,Xiaoaiping, Fluorouracil,Xiaoaiping combined with Fluorouracil,Mitomycin,Xiaoaiping combinedwith Mitomycin,Survived5,7,5,5,5,7,Xiaoaiping,Fluorouracil,Xiaoaip ing combined with Fluorouracil,,Mitomycin,Xiaoaiping combined with Mitomycin,Each inhibition rate was 22.6%,22.6%,60.8%,37.4%, 42.9%,Physiological saline group of tumor weighs 9.72 + 0.47 g,Xiaoaiping group of tumor weighs 7.52 + 0.39 g,Fluorouracil group of tumor weighs 3.80 + 0.95 g, Xiaoaiping combined with Fluorouracil group of tumor weighs 2.65 + 0.79 g,Mitomycin group of tumor weighs6.08+ 0.42 g, Xiaoaiping combined with Mitomycin group of tumor weighs5.54+0.41 g,The results show:Physiological saline group and the control group is statistically significant,Mitomycin group and Xiaoaiping combined with Mitomycin have no significant difference,Fluorouracil,Xiaoaiping combined with Fluorouracil and Xiaoaiping also have significant difference.5 VEG immunohistochemical:Saline,Xiaoaiping,Fluorouracil, Xiaoaiping combined with Fluorouracil,Mitomycin,Xiaoaiping combined with Mitomycin,Strong positive rates were:80.0%,57.1%,40%,20%,40%, 42.8%,No significant difference by chi-square test.Conclusion:1 Xiaoaiping had a significant inhibitory effect on Lewis lung carcinoma transplanted tumor.2 Xiaoaiping make tumor cells mainly stay in the G1 phase,increased proportion of cells in G0 / G1 phase,significantly reduced the proportion of cells in S phase.3 Xiaoaiping cell cycle phase-specific joint g1 mitomycin chemotherapy can improve living conditions in mice,reduces mortality in mice.4 Inhibitory rate results:Xiaoaiping joint G1 stage of cell cycle specificity Mitomycin effective inhibitory rate higher than that of Mitomycin alone, Xiaoaiping joint G1 stage of cell cycle specificity effective tumor suppression effect is better than G1 stage of cell cycle specificity start alone.5 VEGF expression of Physiological saline group is higher than the experimental group,Fluorouracil group,Xiaoaiping combined with Fluorouracil,VEGF expression is lower than the rest of the group.and Xiaoaiping joint S phase of cell cycle specificity combined use of Fluorouracil VEGF expression is lower than in the effective use Fluorouracil group, eliminate Xiaoaiping based on that the aspects related to the inhibition of tumor angiogenesis. |
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