Objective To explore the preparation of liposomal clodronate and investigate its inducing effects on the apoptosis of peritoneal macrophages in rats after severe acute pancreatitis (SAP).Methods Liposomal clodronate was prepared by means of thin film. SAP rat model was established by retrograde injection of 5% sodium taurocholate into the pancreatic duct. The peritoneal macrophages were obtained from SAP rats. After exposure to different doses of liposomal clodronate (50, 100,150 μl), the PM proliferation was determined by MTT colourimetry. The apoptosis of PM was measured by flow cytometry and agarose gel electrophoresis, respectively. Results The prepared liposomal clodronate had a suitable encapsulation efficiency of clodronate (5.8%) with an average size of 200 nm. The spherical shape of liposome was confirmed by transmission electron microscope. Exposed to liposomal clodronate of different doses resulted in a obvious growth depression (P＜0.01). The apoptotic rate of the PM was (10.32±0.34) %, (18.16±0.49)% and (29.87±0.35)% in three different dose groups and the difference was marked (P＜0.01). 1.2% of agarose gel electrophoresis of DNA extracted from apoptotic macrophages induced by liposomal clodronate showed clearer and characteristic ladder following the liposomal clodronate concentration. Conclusion Liposomal clodronate has a definite effect on peritoneal macrophages in SAP rats.

Objective To investigate the apoptosis of Kupffer cell (KC) induced by lipsomal clodronate in rat with acute necrotizing pancreatitis (ANP). Methods Lipsomal clodronate was prepared by means of thin film, the model of ANP was established by injection of 5% sodium taurocholate of 4 ml/kg into the pancreatic capsule. The Kupffer cells were obtained from ANP rat. After exposure to different doses of lipsomal clodronate (0, 50, 100, 150 μl) , then the proliferation and apoptosis of KC was measured by MTT, flow cytometry and agarose gel electrophoresis of DNA. Results The prepared lipsomal clodronate had an average size of 100～200 nm, the spherical shape of liposome was uniform and confirmed by transmission electron microscope. When exposed to different concentration of lipsomal clodronate for 24 h, the growth suppression rate was 17. 4% , 24. 2% and 31. 1% , respectively, while the apoptosis rate of the KC was (14. 12 ±0.37)% , (18.74±0.43)% and (27.51 ±0.39)%, respectively; the difference was statistically significantly (P<0. 01) , the DNA of KC began degradation and gradually showed clear and characteristic ladder. Conclusions Lipsomal clodronate could induce apoptosis and suppress the growth of Kupffer cells in ANP rats.

Objective To establish a model of cervical spondylosis in rats. Methods Sixty SD rats (four months old) were ran-domly divided into control, muscle imbalance and posterior column instability group, ten rats in each group. Then the degeneration of X-ray film and motion function were evaluated by oblique board test. Results After analyzed the cervical films of control groups, the nature curve still existed, vertebral clearance had no abnormal. No ostensis spurs or ossifies in Luschka joint and joint processes were found. But in mus-cle imbalance and posterior column instability groups, the nature curve was disappeared or stiff, vertebral clearance were stenosis, osteosis spur formed, ossify or sclerosis emerged in Lnschka joint and joint process. Compared with control group in oblique board test, the muscle force decreased in muscle imbalance and posterior column instability group. There were no statistic significant difference between 2 and 4 months of control group, and also no difference in muscle imbalance and posterior column instability group, but there were significant differ-ence in 2 and 4 months of muscle imbalance and posterior column group. Conclusion Not only cervical posterior column instability but also muscle imbalance could result in cervical spendylosis, both muscle force balance and posterior column were important factors in maintaining spine stability.

Summary: A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E.coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28 % of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.

Previous studies have shown that oxidative stress and corporal fibrosis in penile tissues of rats were key pathological factors of erectile dysfunction induced by diabetic mellitus (DMED). Lipoxin A4 (LXA4) was reported to inhibit oxidative stress and fibrosis diseases, while whether it could exert a protective role on erectile function was not clear. Type I diabetic mellitus (DM) was induced in thirty male 10-week-old Sprague-Dawley rats using streptozotocin. Ten weeks later, twenty-two rats with DMED confirmed by an apomorphine test were divided into two groups: The DMED group (n = 11) and the DMED + LXA4 group (n = 11; LXA4 injection daily for 4 weeks). In addition, another ten age-matched rats formed the Control group. We found that erectile function was significantly impaired in the DMED group compared with the Control group, but was improved in the DMED + LXA4 group. Similarly, the over-activated oxidative stress and impaired endothelial function in the DMED group were both improved in the DMED + LXA4 group. Moreover, the DMED group showed serious corporal fibrosis, which was also inhibited by the treatment of LXA4 in the DMED + LXA4 group. Taken together, LXA4 could exert an inhibition role on oxidative stress and fibrosis to improve DMED effectively.

Our previous studies have demonstrated that erectile function was preserved in aged transgenic rats (TGR) harboring the human tissue kallikrein 1 (hKLK1), while the molecular level of hKLK1 on corporal fibrosis to inhibit age-related erectile dysfunction (ED) is poorly understood. Male wild-type Sprague-Dawley rats (WTR) and TGR harboring the hKLK1 gene were fed to 4- or 18-month-old and divided into three groups: young WTR (yWTR) as the control, aged WTR (aWTR), and aged TGR (aTGR). Erectile function of all rats was assessed by cavernous nerve electrostimulation method. Masson′s trichrome staining was used to evaluate corporal fibrosis in the corpus cavernosum. We found that the erectile function of rats in the aWTR group was significantly lower than that of other two groups. Masson′s trichrome staining revealed that compared with those of the yWTR and aTGR groups, the ratio of smooth muscle cell (SMC)/collagen (C) was significantly lower in the aWTR group. Immunohistochemistry and Western blotting analysis were performed, and results demonstrated that expression of α-SMA was lower, while expressions of transforming growth factor-β 1 (TGF-β1), RhoA, ROCK1, p-MYPT1, p-LIMK2, and p-cofilin were higher in the aWTR group compared with those in other two groups. However, LIMK2 and cofilin expressions did not differ among three groups. Taken together, these results indicated that the RhoA/ROCK1/LIMK/cofilin pathway may be involved in the corporal fibrosis caused by advanced age, and hKLK1 may reduce this corporal fibrosis by inhibiting the activation of this pathway to ameliorate age-related ED.

The aim of this study was to investigate the mechanism by which a diet inducing high hyperhomocysteinemia (HHcy) leads to the deterioration of erectile function in rats and whether this is inhibited by expression of the human tissue kallikrein-1 (hKLK1) gene. We established a rat model of HHcy by feeding methionine (Met)-rich diets to male Sprague-Dawley (SD) rats. Male wild-type SD rats (WTRs) and transgenic rats harboring the hKLK1 gene (TGRs) were fed a normal diet until 10 weeks of age. Then, 30 WTRs were randomly divided into three groups as follows: the control (n = 10) group, the low-dose (4% Met, n = 10) group, and the high-dose (7% Met, n = 10) group. Another 10 age-matched TGRs were fed the high-dose diet and designated as the TGR+7% Met group. After 30 days, in all four groups, erectile function was measured and penile tissues were harvested to determine oxidative stress, endothelial cell content, and penis fibrosis. Compared with the 7% Met group, the TGR+7% Met group showed diminished HHcy-induced erectile dysfunction (ED), indicating the improvement caused by hKLK1. Regarding corpus cavernosum endothelial cells, hKLK1 preserved endothelial cell-cell junctions and endothelial cell content, and activated protein kinase B/endothelial nitric oxide synthase (Akt/eNOS) signaling. Fibrosis assessment indicated that hKLK1 preserved normal penis structure by inhibiting apoptosis in the corpus cavernosum smooth muscle cells. Taken together, these findings showed that oxidative stress, impaired corpus cavernosum endothelial cells, and severe penis fibrosis were involved in the induction of ED by HHcy in rats, whereas hKLK1 preserved erectile function by inhibiting these pathophysiological changes.

Nonmuscle invasive bladder cancer (NMIBC) is mainly composed of three different types of tumors: papillary urothelial carcinoma is limited to the mucosal layer (Ta), high-grade carcinoma in situ is limited to the epithelial layer (CIS) and tumors invading the submucosa or lamina propria (T1). The standard treatment for NMIBC is complete transurethral resection of bladder tumors (TURBT) with or without intravesical instillation therapies. However, some high-risk patients are at risk of tumor progression and therefore require more aggressive treatment. Studies have reported that delayed cystectomy can lead to a significant reduction in survival benefits. Therefore, for these NMIBC patients who are at high risk of disease progression, when to abandon conservative treatment and choose cystectomy is one of the biggest challenges. This article reviews the current application status and future directions of radical cystectomy as the initial treatment on NMIBC patients.

To investigate the effect of costimulatory factors in the pathogenesis of chronic idiopathic thrombocytopenic purpura (CITP), we examined the expression of CD80 on platelets and megakaryocytes in patients with CITP and the controls by FACS. By using CD80 monoclonal antibody (McAb) to inhibit interaction among cells which is mediated by costimulatory factors, we observed the effect of CD80 McAb on the growth and maturation of megakaryocytic progenitors of patients with CITP in vitro. The results showed the expression of CD80 on platelets and megakaryocytes in CITP group was significantly higher than that in controls (P<0.01). There was a significantly positive correlation between the expression of CD80 on platelets and serum PAIgG in CITP (r=0.86, P<0.05). The mean of various clone numbers (CFU-MK, BFU-MK and mCFU-MK) in CITP were all lower than those in controls (P<0.05). In megakaryocytes co-cultured with CD80 McAb, there was an increasing tendency of the number of CFU-MK and big CFU-MK (the number of megakaryocyte with GP IIIa positive was more than 20) and mediate CFU-MK (the number of megakaryocyte with GP IIIa positive was 11-20). When the concentration of CD80 McAb was 10 microg/L, there was a significant difference in the number of megakaryocytic colony formation (CFU-MK, BFU-MK and mCFU-MK) between the group with CD80 McAb and that without it (P<0.05). These showed the abnormality of costimulatory factors had important effect in the pathogenesis of CITP.
Adult ; Antibodies, Monoclonal ; B7-1 Antigen ; biosynthesis ; Blood Platelets ; metabolism ; Cells, Cultured ; Chronic Disease ; Colony-Stimulating Factors ; metabolism ; Female ; Hematopoietic Stem Cells ; metabolism ; Humans ; Male ; Megakaryocytes ; cytology ; metabolism ; Middle Aged ; Purpura, Thrombocytopenic, Idiopathic ; etiology ; metabolism

To investigate the expression of hypoxia inducible factor-1 alpha (HIF-1alpha) and its relationship to apoptosis and proliferation in laryngeal squamous cell carcinoma (LSCC), immunohistochemical method was used to detect the expression of HIF-1alpha and PCNA. Tunnel technique was used to detect in situ cell apoptosis in LSCC. Our results showed that the expression of HIF-1alpha was related to the clinical stages of cancer and lymph node metastasis (P<0.05). The relationship between HIF-1alpha and PCNA was statistically significant (P<0.05) and no relationship was found between HIF-1alpha and apoptosis (P>0.05) It is concluded that HIF-1alpha plays a role in the carcinogenesis of laryngeal carcinoma and is correlated with proliferation, but bears no relationship with the apoptosis of tumor cells in LSCC.
Adult ; Aged ; Apoptosis ; physiology ; Carcinoma, Squamous Cell ; metabolism ; secondary ; Cell Proliferation ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; genetics ; Laryngeal Neoplasms ; metabolism ; pathology ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Proliferating Cell Nuclear Antigen ; biosynthesis ; genetics ; Tumor Cells, Cultured