| The occurrence,infiltration and metastasis of gastric cancer is a multi-gene,multi-stage,multi-level synthesis and a variety of signal pathway involved in the process.With the development of tumor,the development process of continuous deep understanding and molecular biology theory and technology development,that molecular targeted therapy is expected to improve the status of cancer treatment.CXCL12,also known as stromal cell-derived factor-1,is closely related to the development of tumors.Previous studies suggest that chemokine receptor CXCR4 is the only receptor for CXCL12,both of which play an important role in tumor infiltration,metastasis and drug resistance.However,studies have shown that in a variety of tumor cells CXCR4 and CXCL12 expression is not parallel.A further study was conducted to confirm a sequence of genes with a high degree of similarity to CXCR4 in the conserved amino acid sequence,named CXCR7.Detection of lung cancer,breast cancer,rhabdomyosarcoma,kidney cancer,cervical cancer,esophageal cancer,melanoma and other primary lesions have CXCR7 high expression in normal tissues and benign tumors almost can not detect CXCR7 expression.At present,the study of CXCR7 mainly in lung cancer,breast cancer,rhabdomyosarcoma,kidney cancer,cervical cancer,esophageal cancer,melanoma,prostate cancer and glial and other tumors.There is little relationship between gastric cancer and CXCR7.This study will examine the relationship between CXCR7 and gastric cancer in three aspects:1.To study the expression of CXCR7-CXCL12 biological axis in human gastric cancer,and to analyze the relationship between CXCR7 and gastric cancer,and to analyze the relationship between CXCR7 and gastric cancer.Human gastric cancer cell lines;2.Screening of effective CXCR7-shRNA sequences and construction of CXCR7-shRNA with lentivirus as carrier;transfection of Lv-CXCR7-shRNA into human gastric cancer cell lines from mRNA and protein expression levels,To investigate the effect of CXCR7 on the biological behavior of gastric cancer cells,3 to verify the inhibitory effect of CXCR7-shRNA on differentiation inhibitor(ID-1),and to study the effect of CXCR7-Effect of CXCL12 biological axis regulation ID-1 on transplanted tumor and angiogenesis in gastric cancer.This work is presented in three parts as follows:Part? Expression of CXCR7/CXCL12 in Different Gastric Carcinoma and Its Relationship with Clinicopathological FactorsResearch purposes:To investigate the expression of CXCR7 and CXCL12 in human gastric cancer tissues and normal tissues,and to analyze the relationship between CXCR7 and CXCL12 expression and clinicopathological factors such as peritoneal metastasis,lymph node metastasis and liver metastasis.Meanwhile,CXCR7 and CXCL12 in 4 human gastric cancer cell lines mRNA expression analysis,for the diagnosis and treatment of gastric cancer to provide a theoretical basis for the potential value.Research contents:1,Immunohistochemical method was used to detect the expression of CXCL12 and CXCR7 in gastric cancer tissues and normal control tissues of 103 patients who underwent surgical resection from May 2015 to May 2016 at the First Affiliated Hospital of Jinzhou Medical University.The expression of CXCL12 and CXCR7 was analyzed by semi-quantitative analysis result;2,The expression of CXCR7 mRNA in four kinds of cells was observed by RT-PCR.The expression of CXCR7 mRNA was detected by RT-PCR.The expression of CXCR7 mRNA in four kinds of cells was observed and identified by SNU-1,SGC7901,BGC-823 and NCI-N87;3,The expression of two proteins in the biological axis was analyzed by Spearman correlation analysis based on the expression of CXCR7 and CXCL12 in gastric adenocarcinoma,and the relationship between protein expression and clinicopathological data was analyzed.Research results:1,CXCR7 and CXCL12 in the gastric adenocarcinoma cell cytoplasm and/or cell membrane were strongly stained and medium to strong staining.CXCR7 and CXCL12 were stained and mediumly stained in normal gastric mucosa,irespectively.The staining intensity of CXCR7 and CXCL12 in the two groups was statistically significant(P<0.05);2,The expression of CXCR7 protein was only weakly expressed in 2 normal tissues(2/103),but the positive rate of CXCR7 in gastric cancer tissues was significantly higher than that in normal tissues(P<0.05).CXCR7 protein was found in 89 Positive staining was found in gastric cancer tissues(89/103).Among them,CXCR7 protein positive staining(67/71)in 67 cases of lymph node metastasis,positive staining in 18 cases of liver metastasis(18/19),and 14 The positive staining rate of peritoneal metastasis was 14.4%,and the positive rates of staining were 86.41%,94.37%,94.74%and 100%respectively.There was no significant difference between group C and group D(P = 0.227).There was a significant difference between the other groups(P<0.05).3,CXCL12 protein was expressed in 64 normal tissues(64/103)and the positive staining rate was 62.14%.The positive expression rate of CXCL12 protein in gastric cancer tissues was significantly higher than that in normal tissues(P<0.05).The positive staining rate of CXCL12 protein was 83.50%in 86 cases of gastric cancer tissues(86/103)CXCL12 protein was expressed in 57 cases of lymph node metastasis(57/71)and expressed in 13 cases of liver metastatic carcinoma(13/19).The positive staining rate was 80.28 in 8 cases of peritoneal metastatic carcinoma(8/14)%,68.42%and 57.14%,respectively,and there was significant difference(P<0.05).4,The expression of CXCR7 and CXCL12 in the primary gastric adenocarcinoma was correlated with the expression of tumor(P<0.01)and the depth of invasion(P<0.05;early stage),and lymph nodes(P<0.01)(P<0.01)and lymphatic vessels(P<0.01),but correlated with the location of tumor(P>0.05)and the size of the tumor(P<0.01)0.05).There was no correlation between age(P>0.05)and gender(P>0.05).5,There was a positive correlation between CXCR7 and CXCL12 expression in primary gastric adenocarcinoma,and the expression of CXCR7 and gastric mucosa was positive(r = 0.611,P<0.05).6,CXCR7 mRNA was highly expressed in two of the four cell lines(SGC7901 and BGC-823)and was low in SNU-1 and NCI-N87 cell lines.Analysis conclusions:1,The expression of CXCR7/CXCL12 in gastric carcinoma was significantly higher than that in normal gastric mucosa.The expression of CXCR7/CXCL12 was closely related to the occurrence,proliferation and growth of gastric cancer.2,With the progression of gastric cancer,CXCR7 expression level and gastric cancer primary tumor through the blood and lymph nodes began to transfer,indicating that gastric cancer cells The expression of CXCR7 may promote the metastasis of tumor.3,With the progress and metastasis of gastric cancer,CXCR7/CXCL12 expression gradually increased.Suggesting that CXCR7/CXCL12 expression in the primary gastric adenocarcinoma is consistent,and the two highly positive expression of gastric cancer cells may have a more aggressive and metastatic;the biological axis is also likely to become synergistic gastric cancer,progress(Invasion and metastasis)and presumably the important molecular markers of prognosis.4,In this study,we examined the expression of CXCR7 mRNA in four kinds of gastric cancer cell lines,and confirmed that some cell lines were not expressed or expressed low,while others expressed CXCR7 mRNA.The gastric cancer cell line SGC7901 is capable of overexpressing CXCR7 and can be used as an experimental subject for the next study.Part? Inhibitory effect of lentiviral vector-mediated RNAi silencing on CXCR7 in human gastric cancer cell line SGC7901 Research purposes:The CXCR7-shRNA expression vector mediated by lentivirus was designed and transfected into human gastric cancer cell SGC7901.The biological function of SGC7901 cells was detected by RNA interference technique.The specific expression of CXCR7 gene was detected by RNA interference.For the gastric cancer CXCR7 targeted therapy to open up new ideas.Research contents:1,The CXCR7-shRNA-14 lentiviral vector was constructed and packaged with CXCR7-shRNA-2,CXCR7-shRNA-3 and negative control were observed by fluorescence inversion microscope.The titer of the virus was determined by the endpoint dilution experiment.2,The optimal infection index of lentivirus carrying the gene of interest was detected.3,Three groups of lentivirus venom and control group were transfected into human gastric cancer cell SGC7901 with the best infection index,and the silencing efficiency of expression vector was analyzed.The highest efficiency of the lentivirus virus interference sequence as the object of the subsequent test;4,MTT assay was used to detect the proliferation of SGC7901 cells and the growth curve was drawn.5,The expression of CXCR7 protein was detected by Western blot.6,Transwell invasion assay was used to quantitatively analyze the number of migrated cells and to calculate the inhibitory rate of silencing CXCR7 on human gastric cancer cell invasion.Research results:1,Three groups of lentiviral vectors and negative control were constructed successfully.Titer showed CXCR7-shRNA-1 of 3.28 × 10~8 TU/ml;CXCR7-shRNA-2 was 4.15 × 10~8 TU/ml;CXCR7-shRNA-3 was 3.84 × 10~8 TU/ml;negative control was 2.73 × 10~8 TU/ml,are in line with lentivirus RNAi experimental requirements,which can be used for subsequent transfection experiments.2,Compared with the control group(D group),the expression of CXCR7 mRNA in the A,B and C groups was significantly lower than that in the control group(P<0.05).The difference was statistically significant(P<0.05)(P<0.05),indicating that CXCR7-shRNA-1 group(group A)could significantly inhibit the expression of CXCR7 mRNA in CXCR7-shRNA-1 group(group A),and the difference was statistically significant(P<0.05)CXCR7 mRNA expression,therefore,select the lentiviral vector CXCR7-shRNA-1 as a follow-up experiment;3,The rate of cell proliferation in the experimental group(group A)was significantly decreased after the transfection of the untransfected virus group(group B)with the geometric multiple.The cell proliferation rate in the experimental group(group A)decreased significantly from the 24th day,(P<0.05).It was proved that CXCR7-shRNA-1 could significantly inhibit the expression of CXCR7 gene and slow down the proliferation rate of SGC7901 cells.4,The expression of CXCR7 protein in the experimental group(A group)was significantly lower than that in the control group and the blank group(P<0.05),while the expression of CXCR7-shRNA-1 was significantly lower than that of the control group There was no significant difference between the two groups(P>0.05).5,The number of cells transfected with CXCR7-shRNA-1 was 97.35 ± 7.49,and the number of cells in the blank group(B)was 253.92 ± 26.13,and the experimental group(A)(P<0.05).The cell inhibition rate of the membrane was 61.66%in the experimental group(A),suggesting that when the number of cells in the membrane was significantly lower than that in the blank group(B),the cell inhibition rate in the SGC7901 cells CXCR7 expression inhibition,can significantly inhibit the ability of SGC7901 cells to distant.Analysis conclusions:1,The recombinant lentiviral vector CXCR7-shRNA,which can silence CXCR7 gene,was successfully constructed,and the silencing efficiency was identified and determined.2,Screening the most efficient CXCR7-shRNA-1 group was used as a follow-up experiment.3,Recombinant lentiviral vector CXCR4-shRNA can significantly inhibit the expression and proliferation of CXCR7 mRNA and protein in gastric cancer cell SGC7901.Part? Effect of CXCR7-shRNA on ID-1 on gastric cancer and angiogenesisResearch purposes:The effect of CXCR7-shRNA on the invasion and metastasis of SGC7901 cells was analyzed by establishing the model of orthotopic transplanted tumor of nude mice.The effects of CXCR7-shRNA on apoptosis and tumor microvessel were also discussed.To provide a theoretical basis for clinical application,while the invasion and metastasis of gastric cancer prevention and treatment to provide new ideas.Research contents:1,The animal model of orthotopic transplanted nude mice was established,and the indexes were observed:body weight and ascites;weight and volume of orthotopic transplanted tumor;survival time,survival rate and tumor inhibition rate of nude mice;2,In the experimental group,24 experimental animals were randomly divided into 3 groups(n = 8).Negative control group(group A):SGC7901 group was transfected separately;positive control group(group B):SGC7901 group transfected with empty virus vector;experimental group(group C):SGC7901 transfected with CXCR7-shRNA lentiviral vector group;3,Immunohistochemistry was used to detect the microvessel density of tumor cells by Weidner microvessel counting.4,Western blot was used to detect the expression of CXCR7,ID1 and VEGF.5,Detection of tumor tissue apoptosis by transmission electron microscopy.Research results:1,The tumor formation time of the three groups was generally 8-15 days,the longest was 24 days;the survival time was between 25-44 days;after 14 days of transfection of the recombinant lentivirus CXCR7-shRNA,Palpable texture harder,less active tumor nodules,the size of 2-3 cm,with the tumor nodules continue to expand,transfected 24 days later,in the upper left abdomen of nude mice palpable obvious tumor mass,Tumor nodules in the same group of nude mice were slightly different,but the difference was not significant;2,In the experimental group,tumor nodules appeared in the nude mice.After 18 days of inoculation,the tumor growth rate was slowed down after 26 days of inoculation,and the tumor mass of each organ in the abdominal cavity was small nodules and the number was small.The positive control group was nude mice Nodules appear in about 12 days after inoculation,after 20 days of inoculation can be observed in the abdominal cavity of the various organs of tumor mass,the group of nude mice for abdominal and thoracic anatomy found:the diaphragm,the lung did not observe the tumor nodules,and abdominal Of the peritoneal,mesenteric,liver,kidney,spleen,intestine were obvious tumor tissue,and the shape is different,different sizes;and negative control group nude mice in about 8 days after inoculation,can be observed in the upper left abdominal surgery area texture Soft tumor nodules,accompanied by tumor nodules do not continue to expand,in the 18 days after inoculation in the right upper abdomen of nude mice can be exposed to various organs of the tumor mass,this time,nude mice were thin,activity decreased significantly;3,Histopathological observation of nude mice with intraperitoneal metastases showed that the tumor tissue was oval,the size of between 1.16 mm-5.45 mm,gray,soft texture,cut surface was fish-like.The tumor cells were adenoid structure,shape,size and arrangement order were irregular morphology,tumor cells chromatin and nuclear pulp is more abundant,nuclear deep dye,mitotic like the more common,was invasive growth,poor cell differentiation,There is no clear boundary with the surrounding organization;4,The mice were sacrificed intraperitoneally after 7 days of transfection of recombinant lentivirus CXCR7-shRNA.The average ascites was 26.67 days in the experimental group.The average ascites was 2.45 ml.The average control group showed ascites at 18.54 days.5.89 ml;negative control group on average in 16.29 days began to ascites,ascites average of 6.62 ml.(P<0.01).However,there was no statistical difference between the positive control group and the negative control group(P<0.01),but there was no significant difference between the positive control group and the negative control group(P<0.01),but there was no significant difference between the positive control group and the negative control group P>0.05);5,The incidence of gastric,liver,intestine,mesentery,spleen,kidney and peritoneum in the nude mice were determined as follows:(1)100%,37.5%,37.5%,25%,25%,12.5%and 25%respectively.The tumor formation rates of the stomach,liver,intestine and mesentery,spleen,kidney and peritoneum were 100%,87.5%,87.5%,75%,75%,62.5%and 75%respectively.The tumor formation rate in the negative control group was close to 100%.The tumor nodules were mainly distributed in the stomach,liver,intestine and mesentery,spleen,kidney and peritoneum.Compared with the other two groups,the difference of tumor formation rate was statistically significant(P<0.01).There was no significant difference between the two groups(P>0.05).6,Three groups of nude mice dissection specimens were observed by the naked eye:tumor nodules color gray,texture is more tough,oval to the organ surface prominent,ranging from mung bean to rice grain size,are isolated nodules,tumor nodules Part of the distribution in the mesentery and intestinal tract,a small amount of distribution in the liver,kidneys,spleen,respectively,growth in the hilar.renal pelvis and spleen,but not invaded the real,and some soft texture of the tumor nodules Film,was gray and red,easy to touch the bleeding;7,Analysis of the appearance of the in situ tumor after dissection in three groups of nude mice showed that the tumor nodules were less in the experimental group,while the positive control group and the negative control group had mild nodules and had a hard texture and irregular shape Outstanding;8,The number of metastatic tumors was 8.5,the maximum weight of the tumor was 0.51 g,the average weight of the metastatic tumor was 1.34 g,and the positive control group had the largest peritoneal metastatic tumor.The maximal diameter and the minimum short diameter of the experimental group were 2.06 mm and 1.16 mm respectively.The mean diameter of the tumor was 5.36mm and 2.06mm respectively,the average number of metastatic tumors was 40.25,the maximum weight of the tumor was 1.13g,the average weight of the metastatic tumor was 3.93g,the maximal diameter and the smallest short diameter of the negative control group Respectively,5.45mm and 2.05mm,the average number of metastatic tumors was 42.75,the maximum weight of the tumor was 1.16g,the average weight of metastatic,tumor was 4.20g.There was no significant difference in tumor length,tumor length,diameter and weight between the two groups(P>0.05).There were significant differences between the experimental group and the other two groups in terms of tumor size,number and weight(P<0.01).The tumor inhibition rate was 68.15%in the experimental group and 6.4%in the negative control group,the difference was statistically significant(P<0.05).9,The average survival time of the experimental group was 41.75 days,which was longer than that of the positive control group at 26.5 days and 24.88 days in the negative control group.In the experimental group,nude mice died on the 28th day and survived on the 28th day.The survival rate was 87.5%.In the positive control group,the nude mice died on the 24th day,and the survival rate was 25%on the 28th day.The negative control group was killed on the 23rd day.Day survived only nude mice 1,the survival rate was 12.5%.The survival rate of nude mice in the experimental group was significantly higher than that in the positive control group and the negative control group(P<0.01).The life extension rate of the experimental group and the positive control group was 40.4%and 6.1%higher than that of the negative control group.10,The difference of microvessel density between the groups was statistically significant(P<0.01).The MVD of the experimental group was 11.38 ± 1.25,which was significantly different from that of the negative control group and the positive control group(P<0.05).The MVD of the control group was 20.17 ± 1.33 and 23.73±2.40,MVD was not statistically significant(P>0.05).11,The expression of CXCR7 protein in the experimental group was significantly inhibited(P<0.01),but there was no significant difference between the two groups(P>0.05),the tumor inhibition rate was 72.54%,Indicating that the silencing CXCR7 gene can significantly inhibit the tumor formation and metastasis,the results and the use of tumor volume calculated tumor inhibition rate of 68.15%of the results consistent;12,The ID1 protein level in the experimental group was significantly lower than that in the control group(P>0.05).There was no significant difference between the two groups(P>0.05).The expression of VEGF protein was also significantly different from that of the control group(P<0.05).13,In the experimental group,the rough endoplasmic reticulum in the nude mice showed degranulation due to mild dilation,and the mitochondria were myeloid and the cells contained nuclear fragmentation.These phenomena indicated that the cells in the experimcntal group had been typical The mitochondrial nembrane defects,a large number of ridge disappeared,mitochondrial myeloidization phenomenon,rough endoplasmic reticulum due to a high degree of expansion of the phenomenon of degranulation,cytoplasmic degeneration and necrosis of the phenomenon,indicating that the control group Cells only degeneration and necrosis,and no typical apoptosis occurred.Analysis conclusions:1,In this paper,we established a model of orthotopic transplantation of gastiric cancer in nude mice.2,Recombinant lentiviral vector CXCR7-shRNA can inhibit tlhe growth of ascites,reduce the tumor tissue growth rate,reduce the tumor tissue volule,reduce the weight of tumor tissue,the cancer cells to different abdominal organs of the transfer process to inhibit,in addition to Can significantly reduce the mortality rate,prolong the survival time of nude mice;3,CXCR7-shRNA recombinant lentiviral vector to inhibit the expression of ID-1,partially block tumor angiogenesis,promote tumor cell apoptosis,and ultimately play a role in inhibiting tumor growth and metastasis. |
PDF Full Text Request