ABSTRACT
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.
ABSTRACT
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.
ABSTRACT
Objective@#To clarify the virus hosting status of human cell lines.@*Methods@#The DNA of Epstein-Barr virus (EBV), Hepatitis B virus (HBV), Human pappilomavirus (HPV), Human immunodeficiency virus (HIV), Human T-cell leukemia virus (HTLV) in 135 human cell lines were checked using PCR, and HCV RNA sequences were checked by RT-PCR. The transmission electron microscopy (TEM) was used to examine the virus particles in cells. The high-risk genotypes of HPV were tested by PCR.@*Results@#By PCR assaying, among the 135 human cell lines, four cell lines(Daudi, Raji, EB-3, NCI-BL2009) harbored EBV DNA sequences; two(Hep3B, PLC/PRF/5) harbored HBV DNA sequences; seven (HeLa, HeLa 229, H1HeLa, HeLaS3, SiHa, Caski, CNE-2Z) harbored HPV DNA sequences, including two cell lines (SiHa, Caski) with HPV16, five cell lines(HeLa, HeLa 229, H1HeLa, HeLaS3, CNE-2Z) with HPV18; one cell line(HUT 102) harbored HTLV DNA sequences. No cell line harbored HCV RNA sequences and HIV DNA sequences. No viral particles were observed in the positive cell lines by TEM, but some viral inclusion bodies in certain cell lines.@*Conclusions@#The virus hosting status of human cell lines can be checked by PCR or RT-PCR. The viral DNA sequences were integrated in the cellular genome.
ABSTRACT
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.
ABSTRACT
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 .
ABSTRACT
Objective:To study the dental and skeletal changes after orthodontic treatment for malocclusion patients with rapid maxil-lary and mandibular expansion companied with fixed appliance.Methods:36 patients underwent the treatment with rapid maxillary and mandibular expansion companied with straight wire appliance.Pre-and post-treatment dental casts and lateral cephalometric radiographs were measured and compared.Results:After treatment,the maxillary and mandibular arch width perimeters increased(P 0.05).U1-SN and U1-NA decreased,L1-MP and L1-NB increased(P <0.05).Conclusion:Dental crowding can be solved effectively and occlusion re-lationship can be kept well with rapid maxillary and mandibular expansion companied with straight wire appliance without influence on the vertical skeletal relationship.
ABSTRACT
Objective:To artificially design and express a recombinant protein named as ScFv-pLLO by fusing ScFv gene of Rituximab(C2B8)and dominant antigen epitopes from listeriolysin O(LLO),and studying its anti-tumor activity.Methods:VH and VL gene sequences of C2B8 against CD20 were acquired by searching United States Patent database,and ScFv sequence was constructed by linking VL and VH with a short peptide linker.Two CD4+T cell epitopes from LLO were selected and designed to splice ScFv sequence.The recombinant gene of ScFv-pLLO was cloned into prokaryotic expression vector and purified after induction.The capacity of ScFv-pLLO target-binding to B-cell lymphomas was evaluated by flow cytometry ( FCM ) and co-immunoprecipitation ( Co-IP ) .The effects of ScFv-pLLO on B-cell lymphomas proliferation and apoptosis were detected respectively.The immunogenicity of ScFv-pLLO was assessed by lymphocyte proliferation assay.Results: ScFv-pLLO was successfully expressed.It could bind to different B-cell lymphomas cell lines and obviously inhibit the growth of Raji cells as well as inducing apoptosis.Moreover,ScFv-pLLO was able to stimulate proliferation of spleen lymphocytes of immunized mice.Conclusion: The recombinant protein ScFv-pLLO can target-bind to B-cell lymphomas,and perform inhibitory effect and induce apoptosis on Raji cells that indicate ScFv-pLLO retain the capacity of ScFv derived from monoclonal antibody against CD20.Besides, ScFv-pLLO can induce immune response.This study provides a basis for further research about the role of ScFv-pLLO on simulating tumor cell antigens as well as being tumor vaccine adjuvant.
ABSTRACT
<p><b>OBJECTIVE</b>To evaluate the effect of combined targeting of MEK and PI3K signaling pathways on K-ras mutated non-small cell lung cancer cell line A549 cells and the relevant mechanisms.</p><p><b>METHODS</b>A549 cells were treated with different concentrations of two inhibitors. Growth inhibition was determined by MTT assay. According to the results of MTT test, the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941,0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244+0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244+5.0 µmol/L GDC-0941). The cell cycle and apoptosis were analyzed by flow cytometry. The expression of proteins related to apoptosis was tested with Western blot.</p><p><b>RESULTS</b>Both GDC-0941 and AZD6244 inhibited the cell proliferation. The combination group II led to a stronger growth inhibition. The combination group I showed an antagonistic effect and combination group II showed an additive or synergistic effect. Compared with the control group, the combination group I led to reduced apoptotic rate [(20.70 ± 0.99)% vs. (18.65 ± 0.92 )%, P > 0.05]; Combination group II exhibited enhanced apoptotic rate [(37.85 ± 3.18)% vs. (52.27 ± 4.36)%, P < 0.01]. In addition, in the combination group II, more A549 cells were arrested in G0/G1 phase and decreased S phase (P < 0.01), due to the reduced expressions of CyclinD1 and Cyclin B1, the increased cleaved PARP and the diminished ratio of Bcl-2/Bax.</p><p><b>CONCLUSIONS</b>For single K-ras mutated NSCLC cell line A549 cells, combination of RAS/MEK/ERK and PI3K/AKT/mTOR inhibition showed synergistic effects depending on the drug doses. Double pathways targeted therapy may be beneficial for these patients.</p>