| Background Esophageal carcinoma is the fourth most frequently occurring cancer in human. Compared with other countries, morbidity of esophageal cancer in China keeps high through recent years, making up nearly half of global incidents. Over 477,900 new cases of esophageal carcinoma were reported across the country in 2015, making the disease a severe issue of public health. Esophageal squamous cell carcinoma is the most common clinical type of esophageal cancer, featuring active infiltration and metastasis which contributes to its extremely high mortality. Researches show that multiple factors are involved in the formation and infiltration of esophageal squamous cell carcinoma, among which are macrophages present in esophageal tumor tissues, named tumor associated macrophages(TAMs). TAMs are divided into two distinct subtypes under different tumor micro-environments. One is classically activated macrophages(M1) which inhibit and kill tumor cells. The other exhibits an alternatively activated phenotype(M2), shown by recent researches to participate in infiltration and metastasis in various carcinomas including gastric cancer, liver cancer and ovarian cancer. This thesis work studies the mechanism of TAMs activity in esophageal cancer cells and xenograft models, providing a fundamental andtheoretical basis for targeted therapy of esophageal cancer.Methods 1.Culture human lymphoma U937 monocytes as TAMs, identify their M2 phenotype by measuring expression of CD206 and CD163 antigens via cytochemical labeling; 2.Induce cultured human lymphoma U937 monocytes into monocyte-derived macrophages by IL-4; co-culture monocyte-derived macrophages with esophageal cancer EC9706 cells as experimental group, while single-culture esophageal cancer EC9706 cells as control group. 3..Measure protein expression levels of Vascular endothelial growth factor(VEGF) and VEGF-C in supernatant of both experimental and control group using enzyme-linked immunosorbent assay(ELISA). 4.Measure cellular expression levels of VEGF and VEGF-C proteins in each group with Western Blot; 5. Measure cellular m RNA expression levels of VEGF and VEGF-C in each group with Real Time PCR(RT-PCR). 6.Measure expression levels of VEGF, VEGF-C proteins, CD206 and CD163 in each group of xenograft tissues by immunohistochemical technique. 7. Measure m RNA expression levels of VEGF and VEGF-C in each group of xenograft tissues with in-situ hybridization method. 8.Measure expression levels of CD31 in each group of xenograft tissues by immunohistochemical technique,in order to mark the blood vessel,computed the value of MVD. 9. Measure expression levels of D2-40 in each group of xenograft tissues by immunohistochemical technique,in order to mark the lymphatic,computed the value of LMVD. 10.Statistical analysis:Experimental data are statistically analyzed using SPSS 17.0 to test whether they follow normal distribution. Results are given as mean +- s.d. Comparison of positive rates is performed using Chi-squared test; comparison ofgroup mean is performed using one-sample Pearson association test or two-sample t-test with significant level of 0.05.Results 1. Positive expression rates of CD163 and CD206 in human lymphoma U937 cells are 100%, identifying them as M2 TAMs for further experiments. 2..After 18 hr of induction by IL-4, human lymphoma U937 monocytes change morphology from spherical to polygonal, and begin to grow attaching to the surface. 3. Supersatant VEGF concentration measured by ELISA shows significant difference(P<0.05) between experimental group(99.81+-0.63) and control group(208.40+-24.587). VEGF-C concentration in supernatant is also significantly different(P<0.05) between experimental group(224.85+-29.33) and control group(208.40+-24.587). 4.Western Blot measurements show that VEGF protein expression level in experimental group cells(0.60+-0.04) is significantly higher than in control group(0.19+-0.02), with P<0.05; VEGF-C protein expression level is also significantly higher(P<0.05) in experimental group cells with M2 TAMs(1.13+-0.23) than in control group cells(0.75+-0.12). 5. RT-PCR measurement show that VEGF m RNA level in experimental group is significantly higher than in control group(P<0.05); VEGF-C m RNA level is higher in experimental group than in control group(P<0.05). 7. Both CD206 and CD163 levels in experimental group measured by immunohistochemical method are significantly higher(P<0.05) than in control group(P<=0.05). 8.m RNA levels of both VEGF and VEGF-C measured by in-situ hybridization method are also significantly higher(P<0.05) in experimental group than in control group. 9.CD31 level in experimental group is 29.21+-5.78/HP and in control group is 26.58+-6.53/HP(P<0.05). 10.D2-40 level in experimental group is 12.13+-1.37/HP and in control group is10.09+-1.21/HP(P<0.05).Conclusions 1. Human lymphoma U937 monocytes can act as tumor associated macrophages, promoting esophageal cancer EC9706 cells to secret VEGF and VEGF-C. 2.M2-polarized TAM promote angiogenesis and lymphatic generation in node mice of esophageal xenograft. |
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