Objective To investigate the gene silencing,apoptosis induction and the suppression of proliferation in vivo transfected by UTMD techniques associated with shRNA techniques. Methods The survivin-shRNA expression vector was constructed. Nude mice were randomly arranged into 3 groups:control group, plasmid injection and ultrasound (P + US), P + UTMD group. Histological examination were evaluated. Protein expressions of Survivin and proliferating cell nuclear antigen (PCNA), Bcl-2, Bax,Caspase-3, Ki-67, nucleostemin (NS), p53 were investigated by immunohistochemistry. Results In transplanted tumors experiment, comparing with those in C and P + US groups, protein expressions of PCNA,Ki-67,Bcl-2, Survivin, NS were down-regulated markedly, while those of Bax, Caspase-3 and P53 were up-regulated significantly ( P ＜ 0.05). Conclusions UTMD combined with shRNA technique can induce apoptosis and inhibit proliferation significantly, without causing any apparently adverse effect,representing a new,promising technology that can be used in the tumor gene therapy and research.

Objective:To transfect genes using ultrasound targeted microbubble destruction (UTMD) techniques and observe the effects of RNA interference on cervical cancer (HeLa) cell line in silencing survivin gene and inducing apoptosis. Methods: Recombinant expression plasmid of short hairpin RNA (shRNA) targeting survivin gene was constructed. It was co-treated with microbubbles and transfected to cultured HeLa cells followed by exposure to ultrasound (P+UTMD group). Moreover, blank control group (C), plasmid group (P), ultrasound exposure group (US), plasmid and ultrasound exposure group (P+US), plasmid+ Lipofectamine group (P+L) were used as controls, respectively. Transfection efficacy was evaluated by observing the red fluorescence in the cells by fluorescent microscopy and flow cytometry(FCM). Ultrasound intensity and exposure time were optimized. Cell apoptosis was investigated using flow cytometry analysis, Hoechst staining, and DNA ladder method. Expression of survivin mRNA was assessed by RT-PCR. Results: Restrictive enzyme digestion and sequencing analysis verified that the recombinant plasmid was successfully constructed. UTMD significantly increased gene transfection efficacy in cultured HeLa cells (P<0.01). Gene transfer was the most prominent at ultrasound intensity of 1.0 W/cm2 and exposure time of 3 min (P<0.01). RT-PCR showed that the expression of survivin mRNA in P+UTMD group was inhibited by (83.33±2.73)%. The differences were significant compared with any other groups (P<0.01). FCM analysis showed that the apoptosis ratio in P+UTMD group was significantly increased as compared with other groups (P<0.01). Hoechst staining and DNA ladder showed that apparent apoptosis and DNA ladder were detected only in P+UTMD and P+L groups. Conclusions:UTMD effectively enhances the transfection efficacy of expression plasmid. It is a novel and effective non-viral gene transfer system and has promising foreground. UTMD mediates RNA interference silenced survivin gene and induces significant cell apoptosis, which provides a new method for tumor research and gene therapy.

[ABSTRACT]AIM:ToestablishandevaluateahyperbilirubinemiaandkernicterusmodelinneonatalSDrats. METHODS:Three-day-old SD rats were randomly divided to 7 experimental groups by litter and body weight , and were in-traperitoneally injected with physiological saline (control group), and 6.25μg/g (T1), 12.5μg/g (T2), 25μg/g (T3), 50μg/g (T4), 100μg/g (T5) and 200μg/g (T6) bilirubin, respectively, twice every day for 3 d.All rats were photo-graphed , weighed and killed 12 h after the last injection .The contents of the stomach were drawn and weighed , and the index was calculated .The liver/body weight ratio was determined , the total and unconjugated bilirubin in the serum and total bili-rubin in the brain were calculated , and the contents of ATP and water in the brain were measured .HE and Nissl staining were used to observe the pathological changes .RESULTS:Along with the increase in bilirubin , gradual exacerbation of the general performance of the rats , and yellowish discoloration of the skin and mucous membranes were observed .The degree of the activity gradually reduced , and the weight gain was suppressed .The weight of T6 group showed negative growth , and the 72 h mortality rate was close to 100%.The mortality rate in T4 and T5 groups continued to rise 1 week after injection .Com-pared with control group , the weight of stomach contents and stomach content index in T 3~T5 groups significantly decreased (P<0.01).The liver/body weight ratio in T5 group was significantly higher (P<0.05).The concentrations of serum total and unconjugated bilirubin and brain bilirubin levels in T 1~T5 groups were gradually increased , while the brain water con-tent had no difference among groups .The brain ATP content in T1~T5 groups increased at the beginning and reached its peak in T3 group, but compared with control group , that in T4 group and T5 group significantly reduced (P<0.05).HE re-sults showed that , with the increase in bilirubin concentration , the number of the neurons in the cerebral cortex of the rats de-creased.In T4 group and T5 group, the neuronal structural disorder , cell swelling, nuclear pyknosis, fragmentation and dis-solution, increase in non-homogeneous structure of the material dyed red , and disappearance of nuclear staining were ob-served.Nissl staining showed that , compared with control group , in T1 group and T2 group, the cortical neurons became smaller, Nissl bodies decreased , and cytoplasmic staining changed little .The cortical neuronal tigroid body color became light gradually, neuron cells become small , and Nissl bodies decreased obviously in T 3, T4 and T5 groups.The T4 and T5 rat ce-rebral cortical neurons dissolved or even disappeared .CONCLUSION:Newborn 3-day-old SD rats receiving intraperitoneal injection of bilirubin at doses of 12.5, 25, 50 and 100μg/g, 2 times a day, can induce hyperbilirubinemia , and 50 and 100μg/g can cause bilirubin encephalopathy .

In the present study the nutritional status in 1723 inpatients were evaluated. The over nutrition in male, in the cadre, and in patients with cerebrooardiovascular diseases or with diabetes was significantly higher than that in female, in the staff members, and the patient changing the valve in heart with rheumatism, respectively. The over nutrition in patients with breast and colonrectal cancer was obviously higher than that in stomach carcinoma patients. The sum total of lymphocytes was reduced in middle malnutrition patients. The nutritional status was well compared with the abnormal ratio of hemoglobin and serum protein, and A/G reverse was increased in some middle malnutrition patients. The MAC, MAMC and TSF in all of the malnutrition patients were than that of 80% standarized value, and were all the same in the evaluation of body weight.

Objective To evaluate the long-term outcome of fully covered self-expandable metal stents(FCSEMS)for the treatment of benign biliary strictures(BBS). Methods Between June 2008 and September 2013, 68 patients with BBS receiving endoscopic retrograde cholangiopancreatography and FCSEMS placement were retrospectively enrolled. Data of endoscopic treatment, stricture resolution and recurrence were collected, and related risk factors were analyzed. Results FCSEMSs were successfully placed in all patients and removed in 93.4%(57/61). The median stent duration was 9.0(range 0.2-37.1)months. Stricture resolution occurred in 74.2%(46/62)patients. During median follow-up of 54.0 (range 2.5-96.0)months,stricture recurrences were seen in 16.7%(6/36)patients. Multivariate analysis revealed that distance between stricture and hepatic bifurcation of less than 1.5 cm(P=0.034,OR=6.395, 95%CI:1.153-35.464), and stent migration(P= 0.024, OR= 0.153, 95%CI:0.030-0.782)were significant risk factors for stricture resolution. Meanwhile, the stricture length longer than 1.0 cm(P=0.028, HR = 6.766, 95% CI:1.233-37.122)was a significant risk factor for stricture recurrence. Conclusion Endoscopic treatment combined with FCSEMS can achieve excellent efficacy in resolving BBS with low recurrence rate. However, location and length of BBS, as well as stent migration may impair its effectiveness.

0.05 ). After once injection both observers considered the number of clearly recognized endocardial border segments increased significantly. The number evaluated by observers A increased from 2.68 ? 0.95 to 5.99 ? 0.10 while from 2.82 ? 1.03 to 5.99 ? 0.11 by observers B( P 0.05 ). The average contrast enhancement rate of LV endocardial border was 99.7 %. Perfluoropropane-albumin microsphere injection had no significant effection on vital signs such as blood prssure, heart rate and respiration. Electrocardiogram didn′t change markedly and the variance of the laboratory findings like blood and urine routine examination, hepatic and renal function was in normal range. Only one case( 0.33 %) had slight side-effects who suffered from mild nausea and diarrhea, which suggested the clinical safety of this contrast agent. Conclusions Perfluoropropane-albumin microsphere injection could enhance the resolution of LV endocardial borders and make the judgement of regional myocardial movement easier. It has little side-effects and will be appropriate for clinical use.

Overlap extension PCR is a common method for site-directed mutagenesis. As objective gene sequence growing longer, it is often difficult to obtain the target product in the second round of PCR, and it is highly possible to introduce unexpected mutations into a long gene fragment by PCR. To circumvent these problems, we can only amplify a small gene fragment which contain the target mutation by overlap extension PCR, and then ligate it with vector to get target plasmid. If the restriction site at the end of the amplified fragment was not a single one on plasmid vector, double fragments ligation method could be used to construct target plasmid. Partial amplification, combined with double fragments ligation, could solve lots of problems in long gene mutagenesis. Taking retinoblastoma gene 1 S780E mutagenesis as an example, it is difficult to amplify whole retinoblastoma gene 1 by overlap extension PCR because of long fragment interfering the overlapping extension of second round PCR. However, it is relatively easy to amplify the F3 (1 968-2 787) fragment which contains target mutation S780E. There is a Nhe I site which can be used for ligation on 5' end of F3 fragment, but another Nhe I site on the plasmid restrained from doing so directly. In order to circumvent this obstacle, we ligated F3 fragment, combining with F2 (900-1 968) fragment which was digested from wild type plasmid, with the vector which contain F1 (1-900) fragment of the gene. That double fragments ligated with one vector at the same time, though less efficient, can recombine into a complete plasmid. The sequences of the two selected recombinant plasmids were consistent with the target mutation, which verified the feasibility of this scheme. As an improvement of overlap extension PCR, partial amplification and double fragments ligation methods could provide solutions for site directed mutagenesis of many long genes.
Base Sequence ; Cloning, Molecular ; Genetic Vectors ; genetics ; Mutagenesis, Site-Directed ; methods ; Nucleic Acid Amplification Techniques ; Plasmids ; Polymerase Chain Reaction

Osteoporotic vertebral compression fracture (OVCF) can lead to lower back pain and may be even accompanied by scoliosis, neurological dysfunction and other complications, which will affect the daily activities and life quality of patients. Vertebral augmentation is an effective treatment method for OVCF, but it cannot correct unbalance of bone metabolism or improve the osteoporotic status, causing complications like lower back pain, limited spinal activities and vertebral refracture. The post-operative systematic and standardized rehabilitation treatments can improve curative effect and therapeutic efficacy of anti-osteoporosis, reduce risk of vertebral refracture, increase patient compliance and improve quality of life. Since there still lack relevant clinical treatment guidelines for postoperative rehabilitation treatments following vertebral augmentation for OVCF, the current treatments are varied with uneven therapeutic effect. In order to standardize the postoperative rehabilitation treatment, the Spine Trauma Group of the Orthopedic Branch of Chinese Medical Doctor Association organized relevant experts to refer to relevant literature and develop the "Guideline for postoperative rehabilitation treatment following vertebral augmentation for osteoporotic vertebral compression fracture (2022 version)" based on the clinical guidelines published by the American Academy of Orthopedic Surgeons (AAOS) as well as on the principles of scientificity, practicality and advancement. The guideline provided evidence-based recommendations on 10 important issues related to postoperative rehabilitation treatments of OVCF.

Neoadjuvant chemotherapy has become an indispensable weapon against high-risk resectable cancers, which benefits from tumor downstaging. However, the utility of chemotherapeutics alone as a neoadjuvant agent is incapable of generating durable therapeutic benefits to prevent postsurgical tumor metastasis and recurrence. Herein, a tactical nanomissile (TALE), equipped with a guidance system (PD-L1 monoclonal antibody), ammunition (mitoxantrone, Mit), and projectile bodies (tertiary amines modified azobenzene derivatives), is designed as a neoadjuvant chemo-immunotherapy setting, which aims at targeting tumor cells, and fast-releasing Mit owing to the intracellular azoreductase, thereby inducing immunogenic tumor cells death, and forming an in situ tumor vaccine containing damage-associated molecular patterns and multiple tumor antigen epitopes to mobilize the immune system. The formed in situ tumor vaccine can recruit and activate antigen-presenting cells, and ultimately increase the infiltration of CD8⁺ T cells while reversing the immunosuppression microenvironment. Moreover, this approach provokes a robust systemic immune response and immunological memory, as evidenced by preventing 83.3% of mice from postsurgical metastasis or recurrence in the B16-F10 tumor mouse model. Collectively, our results highlight the potential of TALE as a neoadjuvant chemo-immunotherapy paradigm that can not only debulk tumors but generate a long-term immunosurveillance to maximize the durable benefits of neoadjuvant chemotherapy.