ABSTRACT
When gene therapy is performed for the treatment of malignant tumors, gene transfer efficiency and selectivity are highly important. Polymer vehicle microspheres are a novel type of therapy, which have been developed rapidly in recent years and are able to control drug release, prolong the biological half-life of drugs, decrease side effects and achieve targeted delivery. The present study was designed to construct a polymer microsphere-encapsulated recombinant adenovirus with human tissue inhibitors of the matrix metalloproteinase-1 (TIMP-1) gene, and to discuss its characterization for the purpose of liver cancer gene therapy. The microsphere was prepared from biodegradable poly-DL-lactide-poly(ethylene glycol) (PELA) encapsulating rAdTIMP-1, the recombinant adenovirus carrying TIMP-1, by a modified double-emulsion method. The particle morphology, diameter, virus encapsulation, loading rate and release kinetics of the rAd-microspheres were determined in vitro. Hepatocellular carcinoma (HCC) HepG2 cells were transfected with the rAd-microsphere and the efficiency of transfection was assessed by fluorescent microscopy. The production and expression of TIMP-1 was identified by gelatin zymography and western blot analysis, and the invasiveness was detected by a matrigel matrix invasion assay. The microsphere encapsulating rAdTIMP-1 was successfully constructed with a diameter of 1.965 µm, encapsulation efficiency of 60.0%, a viral load of 10.5 x 10(8)/mg, a virus release of ~60% within 120 h and a total release time of >240 h. The resultant rAd-microspheres were able to efficiently transfect HepG2 cells with the transfection efficiency enhanced by ~90%. As a result, the transfected HepG2 cells had significantly increased TIMP-1 enzyme activity and the expression of TIMP-1 was detected by western blot analysis. In addition, the proliferation and invasion ability of the HCC cells was markedly inhibited by the rAd-microspheres. The resultant rAd-microspheres, PELA-encapsulated recombinant TIMP-1 adenovirus, had enhanced transfection efficiency and were able to markedly inhibit the in vitro biological behavior of HepG2 cells. This provides an experimental basis for this polymer application and may pave the way for prospective in vivo clinical trials and further comprehensive therapy for liver cancer.
Subject(s)
Adenoviridae/genetics , Genetic Therapy/methods , Lactates/chemistry , Polyethylene Glycols/chemistry , Tissue Inhibitor of Metalloproteinase-1/genetics , Transfection/methods , Adenoviridae/chemistry , Animals , Cell Movement , Cell Proliferation , Collagen/chemistry , Drug Combinations , Drug Compounding/methods , Gene Expression , Genetic Vectors , Hep G2 Cells , Humans , Laminin/chemistry , Microspheres , Particle Size , Proteoglycans/chemistry , Rats , Tissue Inhibitor of Metalloproteinase-1/metabolism , TransgenesABSTRACT
AIM: To investigate the efficacy of tandospirone in patients with irritable bowel syndrome-diarrhea (IBS-D) and anxiety in a prospective, randomized, controlled study. METHODS: Two hundred patients with IBS-D and moderate anxiety were randomized to receive pinaverium and tandospirone (arm A) or pinaverium and placebo (arm B). Tandospirone or placebo was given thrice daily at a fixed dose of 10 mg and pinaverium was given thrice daily at a fixed dose of 50 mg. The duration of treatment was 8 wk. Patients were assessed for abdominal pain and diarrhea. Anxiety was evaluated using the Hamilton Rating Scale for Anxiety (HAM-A). The primary study endpoints were response rates for abdominal pain and diarrhea. The secondary study endpoints were response rates for anxiety. Adverse events were also evaluated. RESULTS: One hundred and seventy of 200 patients (82 patients in arm A and 88 patients in arm B) completed the study. Demographic and baseline characteristics of the 200 participants were comparable in the two arms. At week 8, the overall response rate for abdominal pain and diarrhea was 52.0% for arm A and 37.0% for arm B (P < 0.05). The HAM-A score showed that the response rate was 61.0% for arm A and 21.0% for arm B (P < 0.01). The treatments were well tolerated and no significant adverse events were reported. CONCLUSION: Tandospirone is effective and can be combined with pinaverium in IBS-D patients with anxiety.
Subject(s)
Anti-Anxiety Agents/therapeutic use , Anxiety/drug therapy , Diarrhea/drug therapy , Irritable Bowel Syndrome/drug therapy , Isoindoles/therapeutic use , Piperazines/therapeutic use , Pyrimidines/therapeutic use , Abdominal Pain/drug therapy , Abdominal Pain/etiology , Adult , Aged , Anti-Anxiety Agents/administration & dosage , Anti-Anxiety Agents/adverse effects , Anxiety/diagnosis , Anxiety/etiology , Anxiety/psychology , Calcium Channel Blockers/therapeutic use , China , Diarrhea/diagnosis , Diarrhea/etiology , Drug Administration Schedule , Female , Humans , Irritable Bowel Syndrome/complications , Irritable Bowel Syndrome/diagnosis , Irritable Bowel Syndrome/psychology , Isoindoles/administration & dosage , Isoindoles/adverse effects , Male , Middle Aged , Morpholines/therapeutic use , Pain Measurement , Piperazines/administration & dosage , Piperazines/adverse effects , Prospective Studies , Pyrimidines/administration & dosage , Pyrimidines/adverse effects , Single-Blind Method , Surveys and Questionnaires , Time Factors , Treatment Outcome , Young AdultABSTRACT
AIM: To investigate the dynamic changes and significance of platelet activating factor receptor (PAF-R) mRNA and protein in pancreatic tissues of rats with severe acute pancreatitis (SAP) and effects of BN52021 (Ginkgolide B). METHODS: Wistar male rats were randomly assigned to the negative control group (NC group), SAP model group (SAP group), and BN52051-remedy group (BN group), and each of the groups was divided into 6 subgroups at different time points after operation (1 h, 2 h, 3 h, 6 h, 12 h, and 24 h) (n = 10 in each). PT-PCR and Western blot methods were used to detect PAF-RmRNA and protein expression in pancreatic tissues of rats respectively. Pathological examination of pancreatic tissues was performed and the serum amylase change was detected. RESULTS: Serum amylase and pathological results showed the that SAP model was successfully prepared, BN52021 was able to decrease serum amylase, and the pathological ratings in BN group at 3 h, 6 h, and 12 h significantly decreased compared with those in the SAP group (8.85 +/- 0.39 vs 5.95 +/- 0.19, 9.15 +/- 0.55 vs 5.55 +/- 0.36, 10.10 +/- 0.65 vs 6.72 +/- 0.30, P < 0.05). The result of PAF-mRNA showed dynamic changes in SAP and BN groups, which increased gradually in early stage, reached a peak at 3 h (0.71 +/- 0.14 vs 0.54 +/- 0.14, 0.69 +/- 0.13 vs 0.59 +/- 0.04, P < 0.05), and decreased gradually later. There were significant differences at each time point except 1 h and 2 h, when compared with those in the NC group (0.71 +/- 0.14 or 0.69 +/- 0.13 vs 0.47 +/- 0.10, 0.38 +/- 0.08 or 0.59 +/- 0.04 vs 0.47 +/- 0.09, 0.25 +/- 0.07 or 0.29 +/- 0.05 vs 0.46 +/- 0.10, 0.20 +/- 0.06 or 0.20 +/- 0.04 vs 0.43 +/- 0.09, P < 0.05), whereas there was no significant difference between BN and SAP groups at each time point. The result of PAF-R protein showed that the change of PAF-R protein in the SAP group and the BN group was consistent with that of PAF-R mRNA. There were significant differences at each time point except 1 h, when compared with those in the NC group (0.90 +/- 0.02 or 0.80 +/- 0.05 vs 0.48 +/- 0.02, 1.69 +/- 0.06 or 1.58 +/- 0.02 vs 0.48 +/- 0.03, 1.12 +/-0.10 or 0.98 +/- 0.03 vs 0.49 +/- 0.09, 1.04 +/- 0.14 or 0.87 +/- 0.02 vs 0.52 +/- 0.08, 0.97 +/- 0.16 or 0.90 +/- 0.05 vs 0.49 +/- 0.10, P < 0.05), whereas there was no significant difference between the BN group and the SAP group. CONCLUSION: PAF-R plays an important role in occurrence and development of SAP. BN52021 exerts biological effects through competitively inhibiting the binding of increased both PAF and PAF-R expression rather than through decreasing PAF-R expression in pancreatic tissues.
