[Establishment of Cas9 stably expressed human hepatocellular carcinoma and cholangiocarcinoma cell lines].

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.

[Specific cytotoxicity of a novel HER2-based chimeric antigen receptor modified T lymphocytes against HER2-positive tumor cells].

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.

[Establishment and validation of human cancer cell lines with stable Cas9 expression].

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.

[Establishment and characterization of a laryngeal squamous cell carcinoma cell line].

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.

Synaptic transmission in simple motility reflex pathways excited by distension in guinea pig distal colon.

We examined specific receptor/transmitter combinations used at functionally identified synapses in ascending and descending reflex pathways of guinea pig distal colon. Excitatory (EJPs) or inhibitory junction potentials (IJPs) were recorded intracellularly from nicardipine-paralyzed circular smooth muscle in either the oral or anal recording chamber of a three-chambered organ bath, respectively. Blockade of synaptic transmission in the central chamber with a 0.25 mM Ca2+/12 mM Mg2+ solution abolished EJPs evoked by distension applied either in the central or the far (anal) chamber. IJPs evoked by distension in the central or the far (oral) chamber were depressed to approximately 50% of control. Hexamethonium (nicotinic receptor antagonist, 200 microM) in the central chamber reduced IJPs evoked by far or central distension to 50%, whereas EJPs evoked by far distension were abolished and EJPs evoked by central distension were reduced to 70% of control. Hexamethonium in the recording chambers reduced both IJPs and EJPs evoked by central distension to approximately 50%. EJPs in the ascending pathway were unaffected by blockade of muscarinic receptors in the central chamber or blockade of neurokinin 3 tachykinin receptors in this or the recording chamber. In the descending pathway, blockade of P2 receptors in the same chambers had only a minor effect on distension-evoked IJPs. Thus some intrinsic sensory neurons of guinea pig colon have long descending projections (>30 mm), but ascending projections of <15 mm. In contrast to the ileum, transmission between ascending or descending interneurons and from sensory neurons to descending interneurons is predominantly via nicotinic receptors; but transmission to inhibitory or excitatory motoneurons and from sensory neurons to ascending interneurons involves nicotinic and other unidentified receptors.

Nicotinic transmission at functionally distinct synapses in descending reflex pathways of the rat colon.

We examined descending reflex pathways in the rat colon using intracellular recording techniques. Inhibitory junction potentials (IJPs) were recorded from circular smooth muscle when descending pathways were excited by combined mucosal compression and distension. IJPs were reduced to 71% of control when synaptic transmission was blocked in the oral stimulation chamber of a divided organ bath suggesting that two reflex pathways exist, the one involving descending sensory neurones and the other involving descending interneurones. Hexamethonium (200 micromol L(-1)) in the recording chamber abolished reflexly evoked IJPs, while in the stimulation chamber, it was as effective as synaptic blockade. When hexamethonium was added to a chamber lying between the stimulation and recording chambers, it again sharply depressed IJPs to 27% of control; an extent similar to synaptic blockade. A P2 receptor antagonist did not reveal any purinergic neurotransmission. Either granisetron (5-HT3 receptor antagonist, 1 micromol L(-1)) or SB204070 (5-HT4 receptor antagonist, 1 micromol L(-1)) in the stimulation chamber significantly decreased IJPs; these decreases were not additive. We conclude that some sensory neurones and interneurones in rat colon have long anally projecting axons and that acetylcholine, acting via nicotinic receptors, is the primary neurotransmitter from sensory neurones, to inhibitory motor neurones and between interneurones.

Evidence for functional NK1-tachykinin receptors on motor neurones supplying the circular muscle of guinea-pig small and large intestine.

The guinea-pig intestine was investigated to determine which neurones are excited via NK1 receptors. The specific NK1 receptor agonists [Sar9, Met(O2)11]-SP and septide contracted the circular muscle of all regions via a tetrodotoxin (TTX)-insensitive mechanism. In the proximal colon, they also evoked a TTX-sensitive relaxation; in the distal colon, the contractions were larger when nerve impulses were blocked with TTX, indicating that the agonists excited inhibitory motor neurones. In the duodenum and ileum, TTX reduced agonist-evoked contractions indicating that excitatory motor neurones were activated. In the presence of indomethacin, TTX enhanced contractions of ileal circular muscle evoked by these agonists suggesting that NK1 receptors were on inhibitory motor neurones. Blockade of nitric oxide synthase (NOS) enhanced NK1 receptor agonist evoked contractions of duodenal circular muscle, indicating that the agonists excite inhibitory motor neurones in duodenum. Neurones immunoreactive for NK1 receptors were studied in the duodenum and distal colon. As reported previously for the ileum, 1 some neurones were immunoreactive for NOS and had Dogiel type I morphology; features characteristic of inhibitory motor neurones. In conclusion, there are functional NK1 receptors on excitatory and inhibitory motor neurones in the guinea-pig small intestine and on inhibitory motor neurones in the colon.