| Gastric cancer is one of the most familiar malignant cancers in the digestive tracts,and its mortality ratio is the primacy in all malignant cancers.The basic and preventive researches have made great progress recently following the rapid developments of molecular biology,immunology,cell biology and pathology.But the most of these researches come from clinical retrospections to the patients with gastric cancer,so there are much localization of the time and space.If we would like to verify the effects of carcinogens,illustrate the mechanisms of gastric carcinogenesis,and discuss the role of anti-tumor drugs,animal models of gastric carcinoma must be established.Human diseased animal models have been playing great roles in medical development.The carcinogens have been used to establish tumor animal models for many years,but its application has been limited because of its disadvantages,such as uncertainty of inducement,randomicity of carcinogenesis,and no biological effects in the whole level.Transgenic animal models are able to overcome these shortcomings mentioned above,the biological effects of transfered genes can be observed in animal whole level.The tissue specific expression and controlling process can be studied. The transgenic animal models have provided valuable data for the researches of functionary mechanisms of oncogene,carcinogenesis and developing process of cancer cells,gene expression and regulation.The transgenic models have initiated a new scientific research approach for animal models of human diseases. Transgenic animal is such an animal -that foreign gene has been injected into its germ cells or embryo,inserted in its chromosome genome and can be transfered to its offspring stably.Transgenic technique is one of the greatest progresses in biology field and is in possession of the infinite foregrounds in the researches of medicine, molecular biology,molecular genetics,animal and veterinary science.At the present time,the foreign genes are often transferred into zygotes by microinjection or electricity transfer.The operation includes the construction of recombinant,collection of zygotes,microinjection of foreign genes,transplantment of injected zygotes and detection of transgenic animals.Transgenic animal models of human genetic diseases and human oncogene have been built abroad,and they have been used in cancer and genetic research.But the study in this aspect in our country is still in initial stage.SV40 belongs to polyomavirus and papovaviridae,used widely in transgenic animal models.The two transfer proteins,large T antigen(Tag) and small t antigen (tag),had been encoded before virus DNA replication.Tag was a phosphoprotein, which was indispensable to cell transfer and played a decisive role in this process,tag was a non-phosphoprotein and it wasn't necessary to cell transfer,but it could enhance this action.SV40T gene had been used widely in establishment of transgenic tumour animal models.For example,Santarelli had transmitted SV40T gene in mouse cells and constructed mouse breast cancer models,Brister transferred SV40T gene and metallothionein gene fusion plasmid into mouse germ line to establish transgenic mice which produced brain venation papilloma,Hochman constructed eye tumor transgenic mouse models.The gastric parietal cells are the kind of main types in fundic gland.They can synthesize and secrete hydrochloric acid which stimulates the secretion of pancreatin and endocrine cells in gastric intestine tracts.H+/K+ATPase gene is expressed in gastric parietal cells specifically and connect to the synthesis and secretion of hydrochloric acid directly.Gordon had established transgenic animal models specifically expressing human growth hormone(hGH) and intrinsic factor(INF) in gastric parietal cells.This model had made foundation for the research of proliferation, evolvement process in gastric mucous cells.We had constructed the eukaryotic specific expression vector SV40T controlled by H+/K+ATPaseβunit promoter and injected it in mouse zygotes to establish transgenic mice.SV40T gene has been specifically expressed in gastric parietal cells.They would induce the hyperplasia of pre-parietal cells,and pathologic changes in gastric mucosa.These models would be very useful experimental animals for the researches on the molecular mechanisms of gastric carcinogenesis,diagnosis and therapy of gastric cancer,and special drug choice.MethodsPartâ… Construction of specific expression vector of SV40 large T antigen in gastric parietal cell1.Amplification of H+/K+ATPaseβunit promoter.The genome DNA was extracted from mouse liver using hydroxybenzene-chloroform method. H+/K+ATPaseβunit promoter was amplified by polymerase chain reaction(PCR) and its product was named as HK.2.Construction of pMT/HK.The PCR product was purified and ligated to pMT18-T vector.The recombinants were transformed to competent cells DH5α.8 clones were picked from the transformed plate and incubated at 37℃overnight. Plasmid DNA was extracted from the bacteria.Positive recombinants were identified with Xbaâ… and BamHâ… digestion and sequenced.They were named as pMT/HK.3.Construction of pcDNA3.1/HK.1060bp DNA fragment was cleaved from pMT/HK with Xbaâ… and BamHâ… restricted enzyme digestion and connected to pcDNA3.1(-) which had also been digested with Xbaâ… and BamHâ… .The recombinants were transformed to competent cells and clones were picked from the transformed plate.Plasmid DNA was extracted and the positive recombinants were identified with restricted enzymes digestion.They were named as pcDNA3.1/HK.4.Construction of pcDNA3.1/HKSV.2700bp DNA fragment was cleaved from pLITAg(including SV40T gene) with BamHâ… restricted enzymes digestion and ligated to pcDNA3.1/HK which had also been digested with BamHâ… .The recombinants were transformed to competent cells and clones were picked from the transformed plate.Plasmid DNA was extracted and positive recombinants were identified with restricted enzymes digestion.The inserted orientation and sequence of SV40T gene were verified by sequencing.The correct orientation recombinants were named as pcDNA3.1/HKSV.5.Construction of pcDNA3.1/SV.2700bp DNA fragment was cleaved from pLITAg with BamHâ… restricted enzymes digestion and ligated to pcDNA3.1(-) which had also been digested with BamHâ… .Plasmid DNA was extracted and recombinants were identified by restricted enzymes digestion.The inserted orientation was verified by sequencing.The correct orientation recombinants were named as pcDNA3.1/SV.Partâ…¡Establishment of transgenic mice expressing SV40T specifically1.Preparation of transgenic DNA.The plasmid DNA pcDNA3.1/HKSV was extracted and digested with Xbaâ… and Kpnâ… .The reaction products were electrophoresed on 0.7%agarose gel.3.7kb H+/K+ ATPaseβpromoter/SV40T fragment was collected and purified by using agarose extraction kit for DNA microinjection.2.Preparation of ligated males,donative females,pseudopregnant females.The DNA fragments were injected into oocyte pronucleus.Injected eggs were transferred into the oviduct of pseodopregnent females using standard techniques.Partâ…¢Identification and proliferation of transgenic mice expressing SV40T specifically1.Detection of transgenic positive founders and F1 offspring using PCR.The genome DNA was extracted from mouse tails.The transgenic founders and F1 positive mice were detected by PCR.2.Detection of F1 transgenic mice using Southern blot.The genome DNA was extracted from the livers of F1 PCR positive mice and detected by Southern blot.3.Detection of SV40T mRNA.The total RNA was extracted with Trizol reagents from the stomach,esophagus,liver,spleen,kidney,heart,pancreas,lung, duodenum,cecum,jejunum,skeletal muscle and testicle of 68# founder mouse,F1 positive mice and non-transgenic B6 mice.The mRNA expression was measured by RT-PCR.4.Detection of SV40T antigen expression by immunohistochemistry.The tissue sections of stomach,esophagus,liver,spleen,kidney,heart,pancreas,lung, duodenum,cecum,jejunum,skeletal muscle and testicle of F1 positive mice and non-transgenic B6 mice were prepared.SV40T antigen expression was detected by immunohistochemistry.Partâ…£The biological characters of transgenic mice expressing SV40T specifically1.Gastric mucosa observation of transgenic mice.The tissue sections of transgenic mice and non-transgenic mice stomach were prepared and stained with HE.The changes of gastric mucosa morphology were observed and the thickness of gastric mucosa was measured.2.Observation of parietal cell's ultrastructure using transmission electron microscopy The stomachs of transgenic mice and non-transgenic mice were fixed, dehydrated,embedded,polymerized,sliced up and stained.The parietal cell's ultrastructure was observed using transmission electron microscopy.3.Measurement of gastric acidity.Eight wild-type and 8 transgenic mice were denied access to food 24h.Gastric fluid in the stomach was collected after polyrus ligation.The PH of the gastric fluid was measured after centrifugation.4.Detection of cell apoptosis by TUNE1 assays.Stomachs of wild-type and transgenic were embedded in paraffin and sections were prepared from the zymogenic zone.TUNEL assays were performed as described in the method recommended by the kit.5.Statistical analysis.The SPSS 12.0 statistical software was used for statistical analysis of the data.To numerical variable,the data were expressed with(?)±s, the t-test and ANOVA were used to compare these data.The level of significant isα=0.05. ResultsPartâ… Construction of specific expression vector of SV40 large T antigen in gastric parietal cell1.The construction of pMT/HK.H+/K+ATPaseβunit promoter was obtained by PCR using the mouse liver genome DNA and P1,P2 primers.1060bp fragment could be seen in 10g/L agarose gel and its length fitted the anticipation,pMT/HK plasmid was digested with Xbaâ… and BamHâ… ,two DNA fragments(1kb and 2.6kb) could be observed and their length accorded to prospective results.2.The sequencing of H+/K+ ATPaseβuint promoter.The sequencing of pMT/HK showed that the length of H+/K+ ATPaseβpromoter PCR products was 1057bp.The sequencing result was compared with the sequence reported by Robert Levenson using Blast 2 sequences software.Base repeated sequence existed at -501bp to -634bp,and there was 1 to 2 different repeated sequence among different mice or reported sequence,so this region might be polymorphism loci.3.Identification of pcDNA3.1/HKSV.When pcDNA3.1/HKSV was digested with Xbaâ… and BamHâ… ,three DNA bands could be observed.Their length was 1 kb,2.7kb and 5.4kb respectively.When it was digested with Xbaâ… and Kpnâ… ,two DNA bands could be seen.One was 3.7kb fragment,the other was 5.4kb.When pcDNA3.1/HKSV was digested with BamHâ… ,2.7kb and 6.4kb DNA bands could be observed.If it was digested with EcoRâ… ,9.1kb DNA band could be detected.The results of restricted enzymes digestion accorded to anticipation.4.Identification of pcDNA3.1/SV,when pcDNA3.1/HKSV was digested with BamHâ… ,2.7kb and 6.4kb DNA bands could be observed.While pcDNA3.1/SV was digested with BamHâ… ,2.7kb and 5.4kb DNA bands could be seen.When pcDNA3.1 was digested with BamHâ… ,only one 5.4kb DNA band could be detected.All these results also accorded to design.5.Sequencing result of SV40T.pcDNA3.1/SV2 was verified to be the recombinant in which SV40T had inserted into pcDNA3.1 at the correct orientation.SV40T sequence in pcDNA3.1/HKSV was the same as NC001669 completely.Partâ…¡Establishment of transgenic mice expressing SV40T specifically1.The purification of injection DNA.pcDNA3.1/HKSV was digested with Xbaâ… and Kpnâ… .3.7kb DNA fragment was collected and purified with agarose gel extraction kit and dissolved in TE buffer for microinjection.Its concentration should be 3ng/μl.2.Establishment of transgenic mice.150 mice were superovulated and pessaries were observed in 96 mice.2500 zygotes were collected and the number of average superovulation was 26/mouse(2500/96).548 zygotes in which pronucleus were clear were used for microinjection and 422 integral germ cells were transplanted to 16 psuedopregnant mice.77 offspring were born and the success ratio of transplantation was 18.2%.Partâ…¢Identification and proliferation of transgenic mice expressing SV40T specifically1.Detection of transgenic mice using PCR.10 offspring were verified to be positive transgenic mice from 77 young mice by PCR.They were 2#,4#,8#,16#, 24#,51#,57#,61#,68# and 73#.The positive ratio of transgenic mice was 13.0% (10/77).2.Reproduction of founder mice and detection of F1,F2 offspring.One founder female and one B6 male,or one founder male and two B6 females were put in one cage to reproduce.68# female mouse was sterile,and 99 mice were born by other 9 pedigrees.None positive mouse was found in 23 offspring of 8# pedigree.31 mice were proved to be positive by PCR in 2#,4#,16#,24#,51#,57#, 61#,and 73# offspring.The positive rate was 40.8%(31/76).F1 offspring were mated with B6 mice to produce F2.There are 51 mice were born in 4#,16#,24#,51#,57#,73# pedigrees,and 27 mice were proved to be positive by PCR. The positive rate was 52.9%(27/51).The proportion between male mice and female mice in F1 and F2 offspring was round about 1:1. 3.Southern blot result.1～2 F1 transgenic mice which had been measured in 4#,16#,24#,57#,73# pedigree by PCR were sacrificed.Genome DNA was extracted from their livers and digested with BamHâ… overnight.SV40T 618bp PCR products were labeled with Gigoxingenin and hybridized with genome DNA of 4#, 16#,24#,57# and 73#F1 mice.2.7kb and 3.8kb DNA hybridized bands could be observed in these pedigrees,but there was no band in non-transgenic mice.4.RT-PCR detection.The Stomach,esophagus,liver,spleen,kidney,heart, pancreas,lung,duodenum,jejunum,caecum,skeletal muscle and testicle were separated from 68# sterile mouse,F1 transgenic mice and non-transgenic mice and stored at -80℃.The total RNA was extracted from these tissues and subjected to reserve transcription.443bp GAPDH bands could be amplified in every tissue. 272bp specific SV40T mRNA fragments were amplified in the stomach of 4#,16#,24#,57#,73# pedigree's offspring and 68#,but not in non-transgenic mice's stomachs.RT-PCR results of esophagus,duodenum,jejunum,caecum,liver,heart, lung,kidney,spleen,skeletal muscle,testicle from positive mice showed that there was no SV40T 272bp fragment in these tissues.5.Detection of immunohistochemistry.SV40T antigen only existed in the stomachs of F1 transgenic mice,but not in non-transgenic B6 mice stomachs or other tissues of transgenic mice,such as esophagus,duodenum,jejunum,caecum, liver,heart,lung,kidney,spleen,skeletal muscle,testicle etc.Partâ…£The biological characters of transgenic mice expressing SV40T specifically1.Stomach mucosa observation of transgenic mice.In transgenic mice,the thickness of gastric mucosa was increased,gastric pit and fundic gland were elongated.The number of mature parietal cells decreased and the number of progenitor cells increased greatly.2.The thickness change of gastric mucosa in transgenic mice.The Thickness of gastric mucosa in transgenic mice on postnatal day 35,60,120 was increased greatly than that of non-transgenic mice on same postnatal day.The thickness of gastric mucosa in P35,P60,P120 transgenic mice was 393±59μm,495±68μm,715±79μm respectively,while that was 243±47μm,300±62μm,315±56μm in non-transgenic mice.The thickness of gastric mucosa in several transgenic mice pedigrees had no significant difference.3.Observation of parietal cell's ultrastructure using transmission electron microscopy. The volume of parietal cells in transgenic mice was smaller than that in non-transgenic mice.The number of mitochondria was decreased and tubulovesicular system was underdeveloped or scarce in transgenic mice.4.Measurement of gastric acidity.The PH of gastric juice from transgenic mice and non-transgenic mice was 2.06±0.5 and 7.1±0.4 respectively.There was significant difference between them(P＜0.01).5.Detection of cell apoptosis by TUNE1 assays.The apoptotic cells in non-transgenic B6 mice were only observed in the surface mucous cells.But in transgenic mice,the apoptotic cells can be found in fundic glands excepting in the surface mucous cells.The total ratio of cell apoptosis in non-transgenic mice and transgenic mice was 2%and 15%respectively.The ratio of cell apoptosis in fundic glands was 0.5%and 12%respectively.There was significant difference between them(P＜0.01).6.The changes of pathology in transgenic mice gastric mucosa.The gland epithelial cell in transgenic mice increased greatly.In some pedigree,atrophic gastritis,local vesicle expanse,necrosis,and inflammation exudation can be observed and the number of gland decreased.7.Observation of sterile founder mouse 68# female founder grew slower than other founders and was sterile.Its hair was sparse,spirit was weary,abdomen enlarged.68# was killed on 150 days old and polypoid carcinoma could be found in its stomach.Peritoneum carcinoma existed in its abdomen,which diameter was about 8mm.There were no significant changes in esophagus,duodenum,jejunum, caecum,liver,heart,lung,kidney,spleen etc. Conclusions1.The eukaryotic specific expression vector of SV40 large T antigen controlled by H+/K+ATPaseβsubunit promoter had been established.H+/K+ ATPaseβsubunit promoter was analyzed by sequencing and repeated polymorphism loci was found in this region.2.The transgenic mice expressing SV40T gene in gastric parietal cells had been established.The gastric specific expression of transgenic mice had been verified by RT-PCR and immunohistochemistry.The thickness of gastric mucosa in transgenic mice was increased,gastric pit and fundic gland elongated and the number of mature parietal cells decreased greatly.3.The ultrastructure of parietal cells in transgenic mice changed.The number of mitochondria decreased and tubulovesicular system was underdeveloped or scarce in transgenic mice.The secretion of gastric acid was decreased. SV40T-induced pre-parietal proliferation was accompanied by apoptosis. Atrophic gastritis,local vesicle expanse can be observed in some pedigree.4.Gastric polypoid tumour and peritoneum carcinoma had been found in transgenic sterile mouse.The transgenic mice would be useful animal models for the research of gastric cancer. |
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