Clustered regulatory interspaced short palindromic repeats (CRISPR) found in bacteria and archaea genome that contains multiple short repeats loci, provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage. The first inkling of this hot new genetic engineering tool turned up in 1987, when a research team observed an oddly repetitive sequence at one end of a bacterial gene. Now three types of CRISPR/Cas system have been identified: types I, II and III. In the type II CRISPR/Cas9 system, short segments of foreign DNA termed 'spacers' are integrated within the CRISPR genomic loci, transcribed and processed into short CRISPR RNA (crRNA). These crRNAs anneal to trans-activating crRNA (tracrRNA) and direct sequence-specific cleavage in that a double-strand break (DSB) is generated by Cas proteins. Based on these findings, various genetic methods, including gene targeting (Gene disruption), gene insertion, gene correction etc., are being designed to manipulate the genomes of different species at specific loci. Compared with zinc finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), CRISPR/Cas9 is simpler with higher specificity and less toxicity. This review summarizes recent progress, discusses the prospects of CRISPR/Cas9 system, with an emphasis on its structure, principle, applications and potential challenges, and provides a useful reference for researchers who are interested in this new technique.
Bacteria ; CRISPR-Cas Systems ; DNA ; Genetic Engineering ; Genomics ; RNA

Objective To discuss whether potassium sensitivity test(PST) is correlated with PUF in Interstitial Cystitis (IC). Methods The data of 14 IC patients (female 13, male 1) were an-alyzed. The mean age was 48 years (range 35-67 years). The clinical symptoms included urinary fre-quency, urgency, pelvic and peritoneal region pain after bladder filling. All the patients met the diag-nostic criteria of NIDDK for IC. Dilatations by hyponome were performed, medicine including heparin-sodium, lidocaine, NaHCO3 were used by intravesical instillation. PST and the pelvic pain and urgen-cy/frequency patient symptom (PUF) were used for evaluation. The relationship of the PST and PUF was assessed by statistics. Results PST median decreased from 4.0 to 1.0 (P<0.01). PUF medi-an decreased from 27.5 to 13.5(P<0.01). PST was directly correlated with PUF (rs=0. 868, t= 4.418, P= 0.001 before treatment, rs = 0.779, t=4.300, P = 0.001 after treatment). Conclusions PST and PUF are correlated. Both can be used as index in diagnosis, differential diagno-sis, symptom severity and treatment effectiveness evaluation of IC.

Objective: To observe the therapeutic effect of extracorporeal shock wave combined with orthopaedic insole on plantar fasciitis. Methods: A total of 153 plantar with plantar fasciitis were randomly divided into a combined group (n=51), an extracorporeal shock wave group (n=53) and an orthopaedic group (n=49). The combined group received treatment of both extracorporeal shock wave and orthopaedic insole while the extracorporeal shock wave or the orthopaedic group only received the treatment of extracorporeal shock wave or orthopaedic insole. The therapeutic parameters such as visual analogue scale (VAS) scores, continued walking time and thickness of the plantar fascia were monitored before and atf er the treatment for 2 weeks, 1 month and 3 months, respectively. Results: The VAS scores in the 3 groups were all reduced after the treatment compared with the corresponding scores before the therapy (P<0.05). hTe VAS score in the extracorporeal shock wave group was greater than that in the orthopedic group atfer the treatment for 2 weeks. hTe VAS score in the combined group was smaller than that in the orthopedic group atfer the treatment for 2 weeks and 3 months (P<0.05). hTe VAS scores in the orthopedic group and the combined group were smaller than those in the extracorporeal shock wave group after the treatment for 1 month or 3 months (P<0.05). The continued walking time and thickness of the plantar fascia was improved after the treatment (P<0.05). hTe cure rate and total effective rate in the combination group were obviously greater than those in the two other groups. hTe cure rate in the orthopedic group was greater than that in the extracorporeal shock wave group (P<0.05). Conclusion: Extracorporeal shock wave combined with orthopaedic insole therapy is an effective method to treat plantar fasciitis. It is recommended to spread in clinic.

Objective To investigate the clinical management of Spontaneous rupture of hepatocellular carcinoma(SRHCC) . Methods This was a retrospective review of the clinical data of patients with SRHCC who underwent treated in the affiliate hospi‐tal of Luzhou medical college from January 2001 to December 2014 .Results Among 104 patients ,small hepatocellular carcinoma (<5 cm) were found in 11 cases ,and large hepatocellular carcinoma(>5 cm) in 93 cases .Thirty‐one cases which underwent surgi‐cal treatment ,were cured;44 underwent transcatheter arterial embolization (TAE) ,5 cases died of liver function failure;29 cases were treated conservatively ,11 cases died of huge bleeding ,18 cases gave up discharged .Twenty‐two small and medium‐sized SRH‐CC cases underwent hepatectomy survived 1to‐10 years ;8 huge sized SRHCC cases survived 5to‐13 months;one case who under‐went partial filling pressure hemostasis and hepatic artery ligation ,but died of tumor rupture again after 34 days .Sixteen cases un‐derwent TAE were followed up ,14 cases survived 3to‐10 moths ,the survival time of two cases were 3 years and 5 years ,respective‐ly .Conservative treatment group has not been followed up .Conclusion The tumours should be surgical resection as soon as possi‐ble in those whose lesions confined to the liver and may be removed ,systemic condition is good;TAE should be used for other pa‐tients .

Objective This experiment was to identify the influence of recombinant human endostatin (rhES) combined with 5-FU on the growth of a xenografted gastric cancer in nude mouse. Methods After the establishment of the model, mice were divided into 4 groups (six in each) receiving respectively, for consecutive 10 days: NS, 5-FU (10 mg/kg) intraperitoneally; rhES (2 mg/kg); and 5-FU + rhES. On 15th day tumor volume was measured. The expression of VEGF, bFGF, VEGF-C, PCNA, bcl-2 and apoptosis index (AI) were examined. The tumor inhibition rate(TIR) and tumor shrinking rate(TSR) were calculated. Results Tumor volume was (43?2) mm 3 in 5 FU+rhES group,(169?45) mm 3 in 5 FU group, (95?28) mm 3 in rhES group, (1057?114) mm 3 in control group ( P

OBJECTIVE:To investigate the long-term toxicity of repeated intramuscular injection of Escherichia coli O157∶H7 polysaccharide-conjugated vaccine(O157) in rats so as to provide safety evidence for clinical trials.METHODS:A total of 48 SD rats were randomly assigned to receive either 0.5 mL vaccine(containing 25 ?g polysaccharides) (immunization group,n=24) or phosphate buffered solution (PBS,control group,n=24) with the same volume for 3 times at a dose interval of two weeks.Sacrifice of 6 rats in each group were scheduled at 2 weeks after first immunization,and at 1,3,and 5 weeks after the third immunization,respectively for observation and determination of hematological and biochemical parameters,histopathology,specific antibody,myeloid tissue,the tissues in injection sites,etc.RESULTS:Compared with control group,immunization group showed no significant pathological change except the dynamic regular change of some hematological parameters induced by the immunization,and no immunologic system damage was observed.CONCLUSION:Repeated intramuscular injection with O157 vaccine in rats wouldn't cause overt toxicity and local irritation.

AIM To study the effects of rh bFGF on healing quality of chronic gastric ulcer in rats. METHODS Ulcer model induced by acetic acid was established. Rats were treated with rh bFGF ig until ulcer healing and then continually treated for 10 days(30 total treatment days). The effects of rh bFGF on healing quality of chronic gastric ulcer was evaluated not only from gastric mucoal barrier tissue structure but also from gastric mucus barrier. RESULTS rh bFGF( 5 000 ～ 10 000 AU?kg -1 ) increased the thickness of regenerated gastric mucosa, lowed its abnormity degree and made its align state near to normal. rh bFGF( 2 500 ～ 10 000 AU?kg -1 ) increased collagen and capillary density in scar tissue, added mucus quantity, mucopolysaccharide layer's thickness and PGE 2 content on the surface of regenerated gastric mucosa. CONCLUSION rh bFGF has effects of promoting gastric mucosal barrier to mature, increasing the function of gastric mucus barrier and increasing the quality of ulcer healing.

OBJECTIVE:To study the mechanism of action of grape seed proanthocynidins extract(GSPE)in treating acetic acid-induced colitis gravis in rats.METHODS:Rats model with acetic acid-induced colitis was built,the animals were di-vided into normal control group,model group,positive control group and GSPE group(which were subdivided into low,medium and high dose subgroups by intragastric administration).On the8th day of administration of drugs,the changes of myeloperoxidase(MPO),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and malonaldehyde(MDA)in colonic tissue were detected,respectively.RESULTS:The model group had a significantly higher level of MPO content than did the normal control group(P

Objective To study treatment of massive variceal bleeding secondary to localized pancreatitis-associated portal hypertension (MVBPAPH).Methods A retrospective study on the clinical data of patients with MVBPAPH was carried out.Of 24 patients with MVBPAPH,20 had pancreatic pseudocysts.12 were operated after failure of treatment with endovascular intervention for variceal bleeding (including 10 patients with splenectomy and gastric fundus-body peripheral vessels amputation and 2 patients with pancreatic pseudocystogastrostomy).8 patients underwent partial splenic embolization and left gastroepiploic artery embolization.4 patients directly underwent splenectomy and gastric fundus-body peripheral vessels amputation for variceal bleeding.Results Left pleural effusion developed in 5 patients who underwent arterial embolization.Left pleural effusion and lung infection occurred in 2 patients who underwent operation.All patients recovered well and were discharged home.During the follow-up period of 2 to 72 months,no rebleeding occurred in these patients (including 2 patients had passed little interval melena).Gastroscopy re-examination showed that variceal veins were not found in 18 patients.Variceal veins which were detected in the remaining 6 patients were obviously less severe.Conclusion Individualized treatment should be given to patients with MVBPAPH and according to the specific type of pancreatitis and the onset time of any accompanying pseudocyst.

Objective To evaluate the effects of fasudil on DCD hearts .Methods Sixteen domestic ,mixed‐breed male porcine [mean body weight (28 ± 3)kg] were randomly divide into experiment group and control group (n= 8 in each) .Experiment group :animals were sedated ,anesthetized and paralyzed .Mechanical ventilation with room air was provided .After thoroctomy ,cardiac arrest was established by bloodletting ,and then keeps at room temperature for additional 25 minutes (warm ischemia) .Hearts were perfused via the aortic root with fasudil (0 .1 mg/kg) enriched cold Stanford solution for coronary artery flushing ,rapidly excised and the aorta was cannulated .Hearts were subjected to isolated Langendorff perfusion (retrograde perfused with warm oxygenated autologous blood) afterwards ,and then underwent 20 minutes of equilibrium ,immersed in fasudil (0 .5 mg/kg) enriched cold Stan‐ford solution for 2 hours in situ cold preservation .Finally ,all the hearts were resuscitated with the warm oxygenated autologous blood perfusion .Control group :hearts from the animals experienced the same except for fasudil supplement .Left ventricular per‐formances were evaluated .Coronary blood flow ,myocardial infarction volume ,myocardial water content ,and myocardial enzyme were measured .Myocardial electron microscopic examinations were carried out as well .Results All the hearts from both groups were successfully resuscitated ,fasudil significantly decreased water content ,enzyme leakage (P< 0 .01 each versus control group) , and increased coronary blood flow (P< 0 .01 versus control group) .Left ventricular function were better preserved (P< 0 .01 each versus control group) .All hearts lacked severe necrosis as determined by tetrazolium staining ,myocardial infarction volume were decresed in experiment group (P< 0 .01 each versus control group) .Intracellular components retained various types of organelle in both group ,but still ultrastructural alterations in control group were more distinctive than in experiment group .Conclusion (1) Donor heart arrested by exsanguinations and plus 25 minutes warm ischemia ,could still be resuscitated with satisfactory result ;(2) The addition of fasudil to Stanford solution (before excise of graft ,during the cold preservation) ,might alleviate DCD heart I‐R in‐jury ,improving the effect of DCD heart preservation ,and hopefully ,might be a novel arsenal in clinical DCD heart transplantation in the future .