Objective The mouse autologous tumor model H22 is more valuable for tumor immunological-related research.This paper aims to establish mouse hepatic tumor cell line(H22-luc2-tdT)that stably express the tan-dem-dimer tomato(tdTomato)and luciferase genes.Establish an in vivo imaging model of cell line derived trans-planted tumors.Methods Using transplanted H22 tumor tissue,primary culture and continuous passage in vitro were performed to establish a continuous cell line.Cell proliferation,chromosome analysis,organoid culture,tumorigenicity,HE and ICH of aFP,CK7,CK15 were performed to charaterize the cell line.Then the luc2-tdT plasmid was transfected into H22 cells of P22,flow cytometry and in vitro/in vivo imaging were employed to screen and verify fluorescence expression.Mycoplasma detection and species verification of the established cell lines were performed.Results The H22 cells had been continuously passaged over 50 times.The cells of passsge 22(P22)were transplanted subcutaneously and intraperitoneally into C57 and Kunming mice,with a 100%tumor formation.The HE morphology of subcutaneous transplanted tumor were consistent with the original tumor.CK+/AFP+proved that it was of liver cancer origin.The H22 cells were hypo-triploid with a modal number of 40-44 chromosomes and telocentromeres,verifing its mouse origin.The latent phase for in vitro growth of H22 lasted from d0 to d3,while the exponential phaes d3 to d5,and reach plateou at d6.Successful transfection of H22 cells with the luc2-tdT were observed with in vitro/in vivo 100%fluorescence positivity,thus named H22-luc2-tdT.The transplanted tumor tissue of H22 cells could be primarily cultured to form organoids.The detection of Mycoplasma was negative,and its mouse origin confirmed by PCR.Conclusions H22 and H22-luc2-tdT cell lines are established and characterized,which can be used for the establishment and application of in vitro and in vivo liver cancer research and metastatic cell tracking.These cell lines are deposited at and can obtain from the National Biomedical Cell Resource Center(http://www.cellresource.cn).

Objective To establish primary and simian virus 40(SV40)T antigen transformed human vascular en-dothelial cell models,and provide available resources for endothelial research.Methods Human umbilical vein endothelial cells(HUVEC),human umbilical artery endothelial cells(HUAEC),great saphenous vein endothelial cells(GSVEC)and endothelial cells form endometrium and liver tissue were isolated and cultured respectively.Then,the primary endothelial cells were transformed by lentivirus containing SV40 big T and small T antigens,and continuously subcultured in vitro.The expression of CD31 was detected by flow cytometry,species identification-and mycoplasma detection by PCR,and cell identity was identified by STR detection.The transformed ECs were checked for HLA types.Some of them were tested for RNA expression profile and infected by Cas9 lentivirus to es-tablish stable clones.Results Totally 187 cell lines of transformed HUVEC,1 of transformed HUAEC,5 of trans-formed GSVEC,1 of transformed endothelial cells from endometrium and 1 of transformed endothelial cells from liv-er tissue,and 9 monoclonal HUVEC cell lines stably expressing Cas9 protein were established.All the transformed umbilical endothelial cells were CD31 positive ranging from 20%-90%for 20 cases,while for the rest 168 cases the positive rate was more than 90%.RNA expression revealed stable activation of cell proliferation(cell cycle and DNA synthesis).Their species were identified as human origin.The STR results were consistent with those of the primary culture and unique,and there was no mycoplasma contamination.All these cells could be obtained with the sharing services of National Science and Technology Infrastructure,the National Biomedical Cell-line Resource cen-ters(NSTI-BMCR).Conclusions A series of primary and SV40 T antigen transformed human vascular endothelial cell models have been established,which provide a tool for the study of cardiovascular diseases,inflammation,tumors and immune-related diseases.

Objective To establish human and mouse pancreatic cancer cell strains stably expressing Cas9 protein,green fluorescent protein,red fluorescent proteins,luciferase-tdTomato,and to validate the activity of luciferase and gene editing of Cas9 function for pancreatic cancer research using luciferase and CRISPR/Cas9 system.Methods In human pancreatic cancer cells(AsPC-1,CFPAC-1,HPAC,BxPC-3,HS 766T,MIA PaCa-2,PANC-1,and SW 1990),and mouse pancreatic cancer cell(Pan02),the cells were infected with Cas9-expressing plas-mid pLv-EF1α-Cas9m1.1-Puro,and single-cell clones were selected for culture and expansion.After extracting the total protein,Western blot verified the expression level of Cas9;Infected with fluorescent protein expression plasmids pLv-EF1α-EGFP,pLv-EF1α-mCherry,pLv-EF1α-tdTomato,pLv-EF1α-Luc2-tdT,and selected single cell clones stably expressing fluorescent proteins were cultured and amplified under fluorescence microscope.Cas9 stable expression cell line was selected to be infected with pLv-EF1α-Luc2-tdT,and the mono-clonal culture of stable expression of fluorescent proteins was selected for expansion under fluorescence micro-scope.One of the cell lines were selected to be infected with Lv-EF1a-mCherry,and the mCherry-positive cells were sorted out by flow cytometry,and then the guide RNA of mCherry gene was then infected by lentivirus to tar-get the mCherry gene,and after cell expansion,mCherry knockdown was detected by fluorescence microscope observation and flow cytometry;5 BALB/c Nude mice were subcutaneously inoculated with MIA PaCa-2-Luc2-tdT cells(1.0×107/cells each),and imaged in vivo after 36 days.Results 48 human pancreatic cancer cell strains with stable Cas9 expression were screened(including 23 cells expressing Cas9m1.1,25 cells expressing Cas9m1.1-Luc2-tdT),33 pancreatic cancer cell strains with stable expression of fluorescent proteins were screened(8 cells expressing EGFP,7 expressing mCherry,and 9 each expressing Luc2-tdT and tdTomato).Cells expressing mCherry and Cas9 were infected with mCherry gRNA and mCherry was knocked down.In vivo imaging showed that both bioluminescence and fluorescence luminescence were present in MIA PaCa-2 cells ex-pressing Luc2-tdT.Conclusions 33 pancreatic cancer cell strains with stable expression of fluorescent proteins are successfully established,in which the Luc2-tdT-expressing cell strains have luciferase activity;48 pancreatic cancer cell strains with stable expression of Cas9 are successfully established,and the Cas9 protein has gene edi-ting activity,gene editing activity varies depending on the original cell strains.

Objective To investigate the effects of nicorandil on hypoxia-inducible factor (HIF)-1α mRNA and protein in lung tissue of one-lung ventilation.Methods Twenty-four clean New Zealand white rabbits were randomly divided into sham group (group S) (two-lung ventilation+thoracotomy),negative control group (group C) (one-lung ventilation + thoracotomy + saline),nicorandil group (group N) (one-lung ventilation+ thoracotomy+ nicorandil) and antagonist group (group J) (one-lung ventilation + thoracotomy + nicorandil + glibenclanide) equally.The implementation of mechanical ventilation depended on self-made double-lumen endotracheal tube after intravenous induction through ear marginal vein.Intravenous maintenance medicine was infused by trace injection pump after anesthesia induction.The implementation of thoracic surgery was simulated through one-lung and two-lung ventilation by auscultation,bubble test and direct observation.Group S was given anaesthesia only,no one-lung ventilation group S,the other three groups had single lung ventilation,and the drug was injected before the operation.Group N was infused nicorandil 100 ptg· kg-1 · h-1 before the implementation of single lung ventilation for 1 h.Group C was injected with the same amount of normal saline.Group J was intravenous infusion of glibenclamide 75 μg· kg-1 · h-1 and nieorandil 100μg · kg-1 · h-1 the implementation of single lung ventilation for 1 h.Then wet and dry weight ratio(W/D) and superoxide dismutase (SOD) activity were measured after non-ventilatory lung was processed and preserved.The expression of HIF-1α protein of non ventilatory lung tissue was detected by Western-blot in the four groups.The transcription of HIF-1α mRNA was detected by real-time quantitative real-time PCR (qRT-PCR) in all groups.Results W/D in groups C and J were significantly higher compared with that of groups S and N (P＜0.05).The activity of SOD in groups C and J was significantly lower compared with groups S and N (P＜0.05).The expression of HIF1α protein and transcription of HIF-1α mRNA in groups C,N and J were significantly higher than those in group S,and that of group N was significantly higher than those of groups C and J (P＜0.05).Conclsion Nicorandil has a protective effect on the collapse and inflation of non-ventilatory lung in rabbit with one-lung ventilation,reducing oxidative stress by SOD,acting on mito KATP and coming into play by up-regulation of HIF-1α.

Objective: To facilitate using the CRISPR/Cas9 gene editing system in human liver and gallbladder cancer cells, we established Cas9 stably expressed human liver and gallbladder cancer cell lines, and validated the gene editing activity of Cas9. Methods: Human liver cancer cell lines (Huh7, PLC/PRF/5, HepG2, Hep3b, SK-HEP-1 and Li-7), human cholangiocarcinoma cells (RBE) and human gallbladder cancer cells (GBC-SD) were infected with 3 Cas9-expressing lentivirus vectors (pLv-EF1α-Cas9-Flag-Neo, pLv-EF1α-Cas9-Flag-Puro, Cas9m1.1), respectively, and Cas9 stably expressed colonies were screened and selected. We extracted the genomic DNA and protein, validated the stable expression of Cas9 by using genomic polymerase chain reaction (PCR) and western blot. Three of cell lines were further infected with Lv-EF1α-mCherry. Then mCherry positive cells were sorted by flow cytometry and infected with designed guide RNA (gRNA) vectors which targeted mCherry gene. Subsequently the gene editing activity of Cas9 was detected by genomic PCR, fluorescence microscopic observation and flow cytometry analysis. Results: One hundred Cas9-expressing human liver and gallbladder cancer cell lines were selected. Among them, 35 cell lines expressed Cas9-Neo, 25 expressed Cas9-puro, and 40 expressed mutant Cas9 (mCas9). We also established 3 cell lines with stable expression of mCherry (Huh7-mCas9-M, PLC/PRF/5-Cas9-M and SK-HEP-1-Cas9-M). The results of genomic PCR and sequencing showed that by lentiviral infection with 2 types of designed gRNA, the long fragment deletion of mCherry gene was found in these 3 cell lines. Moreover, mCherry^-EGFP⁺ cells infected with 2 types of gRNA were observed by fluorescence microscope. The results of flow cytometry showed that mCherry^-EGFP⁺ cells accounted from 0.3% to 93.6%. Conclusion We successfully establish 100 human liver and gallbladder cancer cell lines with stable expression of Cas9 protein and validate their activities of gene editing.

Objective To efficiently builds up and expand breast cancer cells from cancer tissue and to identify their biological properties , provide abundant materials for research and personalized medicine .Methods Feeder cell layer and ROCK inhibitor Y-27632 were employed to faciliate the breast cancer cells;CCK-8 was used to determine the proliferation of the breast cancer cells; Cell cycle distribution was analyzed by flow cytometry; Histochemistry ( FH) assay to show the expression level of CK .The mRNA expression of HER-2, ER, PR and the breast cancer stem cell associated molecules (such as CD44, CD24, etc.) were detected by RT-PCR;STR assay was used for identifying verification of the cells .Results The use of feeder cells and Y-27632 facilitates rapid expand of the original breast cancer cells , and the cells have kept the original features of the tumor .Conclusions To use the method could obtain a large number of cells within a short time , which can promptly be used for the research of per-sonalized medicine .

Objective: To construct the third generation chimeric antigen receptor based on a novel humanized anti-HER2 H1-2 scFv, and to investigate the specific cytotoxicity of H1-2 CAR modified T lymphocytes(CAR-T) against HER2⁺ tumor cells. Method: The expression cassette of the third generation CAR gene and anti-HER2 H1-2 scFv were constructed and cloned into lentivirus transfer plasmid, and then the third generation H1-2 CAR was transduced into human T lymphocytes using lentivirus.Enzyme linked immunosorbent assay was used to detect the expression of cytokines IL2, and LDH release assay was used to detect the cytotoxic effect of the H1-2 CAR-T.Finally, NOD/SCID mice and HER2⁺ breast cancer cell line SKBR3 were used to detect the anti-tumor effect of H1-2 CAR-T in vivo. Results: The third generation H1-2 CAR was successfully constructed.H1-2 CAR-T secreted high dose of IL2 after confrontation with HER2⁺ breast cancer cells.In vitro, the cytolytic rate of H1-2 CAR-T on high expression HER2⁺ tumor cells was significantly higher than that in low expression HER2 or non-expression HER2 tumor cells. At the efficacy to target ratio of 20, the cytolytic rate of H1-2 CAR-T against breast cancer cell SK-BR-3 could reach (90.1±2.8)%, while the cytolytic rate of H1-2 CAR-T against HER2^- breast cancer cell MDA-MB-231 was only (13.5±4.7)%. In the mouse xenograft tumor model, H1-2 CAR-T cells inhibited breast cancer growth in vivo.At the end of the experiments, the average tumor weight in the H1-2 CAR-T cell treatment group was (0.7±0.1) g, the non-transfected T cell therapeutic group was (1.2±0.2) g, and the PBS group was (1.2±0.2) g. There was significant difference between the H1-2 CAR-T therapeutic group and the non-transfected T cell therapeutic group (P<0.05). However, there was no significant difference between the non-transfected T cell therapeutic group and the PBS treatment group (P>0.05). Conclusion The HER2-sepcific H1-2 CAR-T cells specifically kill HER2 positive cells, and further studies on CAR-T cells for the treatment of HER2⁺ cancers are useful.

Objective To investigate the effects and possible mechanisms of nicorandil on lung injury of the collapsed lung in one-lung ventilation.Methods Twenty-four clean Japanese big-ear rabbits were randomly divided into sham group (group S) (two-lung ventilation + thoracotomy),negative control group (group C) (one-lung ventilation + thoracotomy + saline),nicorandil group (group N) (one-lung ventilation+thoracotomy+nicorandil) and antagonist group (group J) (onelung ventilation+ thoracotomy+ nicorandil+ glyburide) equally.Mechanical ventilation was implemented through self-made double-lumen endotracheal tube after intravenous induction through ear marginal vein.Intravenous maintenance medicine was infused by trace injection pump after anesthesia induction.Thoracic surgery was simulated through one-lung or two-lung ventilation determined by auscultation,bubble test and direct observation.Then wet and dry weight ratio (W/D) and content of MDA were measured after non-ventilatory lung was processed and preserved.The expression of Akt,p-Akt and NF-κB protein in non-ventilatory lung tissue were detected by Western-blot in all groups.Results In respects of W/D and content of MDA,the other three groups had significant differences compared with group S (P ＜ 0.05).It was significantly lower in group N than in group C (P ＜0.05),and it was significantly higher in group J than in group N (P＜0.05).The expressions of pAkt protein and p-Akt/Akt in group N were significantly higher than those in group S and group C (P＜0.05).Those of group J were significantly lower than group N (P＜0.05).Compared with group S,the expression of NF-κB protein in group C was significantly higher (P＜0.05).That of group N was significantly lower than that of group C (P＜0.05).But that in group J was higher than that in group N (P ＜ 0.05).Conclusion Nicorandil has a protective effect on the collapse and inflation of non-ventilatory lung in rabbits under one-lung ventilation,acting on mitoKATP through PI3K/Akt,and down-regulating NF-κB to reduce IR-induced lung injury.

Objective: To establish human cancer cell strains with stable Cas9 expression, and to validate the gene editing activity of Cas9 for simple gene editing in future study. Methods: Fifteen cancer cell lines of different tissue origins were infected with pLv-EF1α-Cas9-Flag-Neo or pLv-EF1α-Cas9-Flag-Puro by lentivirus and clone selection was employed to screen Cas9 stably expressed cancer cell lines. Afterward designed guide RNA vectors targeting TSC22 gene were transiently transfected into 3 of cell lines, and subsequently the gene editing activity of Cas9 was evaluated by genomic PCR, sequencing and Western blot. Results: Sixty-nine human cancer cell strains with stable Cas9 expression from different cancers were established, and by transient transfection with designed guide RNA, long fragment deletion was detected in TSC22 gene. Conclusions Sixty-nine human cancer cell strains are successfully established with stable expression of Cas9 protein and gene editing activity. These cell strains may be employed in large-scale drug screening, screening of new drug targets and gene function investigation.

Objective: To establish a laryngeal squamous cell carcinoma (LSCC) cell line through primary cell culture and observe its biological characteristics. Methods: Tissue block culture method was used for primary cell culture. After LSCC cells passed 25 times in vitro, the morphology of cells was observed, keratin was stained histochemically, cell cycle was tested by PI-FACS, and the specie of cells was detected by PCR and short tandem repeat(STR) typing. Results: This newly established LSCC cell line was named as TR-LCC-1, most of the cancer cells were polygonal shape, like the cobblestone, loss of contact inhibition and with overlapping growth. Cell size was large and cell pleomorphism was very obvious. Cytokeratin staining was positive. After 6 months of continuous culture in vitro, the TR-LCC-1 cells passed more than 30 times, and cell doubling time was 201.2h. Cell cycle assay indicated that G1 phase accounted for 51.71%, S phase was 44.56%, and G2 phase was 2.28%. Mycoplasma test showed no mycoplasma contamination. Cell species identification identified TR-LCC-1 was human-derived cells. STR detection showed P26 and P6 were same, and they were different from the STR typing of disclosed cells. Conclusion The establish ment of the new laryngeal squamous carcinoma cell line TR-LCC-1 can be helpful to the research for laryngeal squamous cell cancer.