BACKGROUNDThe treatment of hypoparathyroidism (HPT) is still a difficult clinical problem, which necessitates a new therapy. Gene therapy of HPT has been valuable, but how to improve the gene transfer efficiency and expression stability is a problem. This study was designed to optimize the gene therapy of HPT with hematopoietic stem cells (HSCs) recombined with the parathyroid hormone (PTH) gene.

METHODSThe human PTH gene was amplified by polymerase chain reaction (PCR) from pcDNA3.1-PTH vectors and inserted into murine stem cell virus (MSCV) vectors with double enzyme digestion (EcoRI and XhoI). The recombinant vectors were transfected into PA317 packaging cell lines by the lipofectin method and screened by G418 selective medium. The condensed recombinant retroviruses were extracted and used to infect HSCs, which were injected into mice suffering from HPT. The change of symptoms and serum levels of PTH and calcium in each group of mice were investigated.

RESULTSThe human PTH gene was inserted into MSCV vectors successfully and the titres were up to 2 x 10(7) colony forming unit (CFU)/ml in condensed retroviral solution. The secretion of PTH reached 15 ng.10(-6).cell(-1) per 48 hours. The wild type viruses were not detected via PCR amplification, so they were safe for use. The mice suffering from HPT recovered quickly and the serum levels of calcium and PTH remained normal for about three months after the HSCs recombined with PTH were injected into them. The therapeutic effect of this method was better than simple recombinant retroviruses injection.

CONCLUSIONSThe recombinant retroviral vectors MSCV-PTH and the high-titre condensed retroviral solution recombined with the PTH gene are obtained. The recombinant retroviral solution could infect HSCs at a high rate of efficiency. The infected HSCs could cure HPT in mice. This method has provided theoretical evidence for the clinical gene therapy of HPT.

Animals ; Antigens, CD34 ; analysis ; Female ; Genetic Therapy ; Genetic Vectors ; genetics ; Hematopoietic Stem Cells ; Humans ; Hypoparathyroidism ; blood ; therapy ; Mice ; Parathyroid Hormone ; blood ; genetics ; Retroviridae ; genetics

OBJECTIVETo investigate the expression features of P-glycoprotein (P-gp), glutathione S transferase-pi (GST-pi) and inhibitor of apoptosis proteins like p53, survivin and bcl-2 in lymph node metastases of gastrointestinal carcinomas.

METHODSThe expression of P-gp, GST-pi, p53, survivin and bcl-2 were determined by using immunohistochemistry technique in surgical specimens of primary tumor (PT) and lymph node metastases (LNMs) from 54 gastrointestinal cancer patients with metastasis of lymph nodes. The expression difference of 5 multi-drug resistance (MDR)-related factors between LNMs and PT were compared.

RESULTSSignificant difference was found in the expression of P-gp and GST-pi between the two groups (both P < 0.05), and expression of p53 and bcl-2 showed positive correlation between LNMs and PT (r = 0.7248, 0.5524; both P < 0.05), respectively. In LNMs, P-gp expression was positively correlated with GST-pi (r = 0.4062, P < 0.05) and survivin (r = 0.6169, P < 0.05), and also GST-pi expression was related positively with survivin (r = 0.4027, P < 0.05). Statistically positive correlations were noted between bcl-2 and P-gp (r = 0.3986, P < 0.05), bcl-2 and survivin (r = 0.2937, P < 0.05), as well as GST-pi and survivin (r = 0.4481, P < 0.01) in PT. Only a positive correlation between GST-pi and survivin expression was simultaneously shown in both LNMs and PT.

CONCLUSIONSThere is significant heterogeneity of MDR-related factors expression in LNMs of gastrointestinal carcinomas. Effective adjuvant chemotherapy after operation should target on the metastatic loci of the disease.

ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Digestive System Neoplasms ; metabolism ; pathology ; Female ; Glutathione S-Transferase pi ; metabolism ; Humans ; Inhibitor of Apoptosis Proteins ; Lymph Nodes ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Microtubule-Associated Proteins ; metabolism ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Tumor Suppressor Protein p53 ; metabolism

OBJECTIVETo study the pathological changes of the liver tissues of patients with HIV infection.

METHODS14 biopsy and 12 autopsy liver tissues were examined histologically. HIV-1 related antigen of outer membrane protein gp120 and capsid protein p24 were examined with their corresponding monoclonal antibodies by immunohistochemistry.

RESULTSIn the biopsy group, cytomegalic virus (CMV) infection was found in one (1/14) case, outer membrane protein gp120 and/or capsid protein p24 antigen were detected in Kupffer cells and in some of the lymphocytes in 11 cases. All the hepatocytes were negative for outer membrane protein gp120 and capsid protein p24 antigens. In the autopsy group, there were 5 (5/12) cases of liver tissues with CMV infection and 5 cases each with mycobacterium and Toxoplasma gondii infection. Capsid protein p24 was detected in liver tissues in 3 cases.

CONCLUSIONThere is HIV infection in liver tissue of patients with HIV. The rate of opportunistic infections in liver biopsy samples was lower than that in the autopsy liver tissues of patients with HIV.

Adult ; Female ; HIV Core Protein p24 ; biosynthesis ; genetics ; HIV Envelope Protein gp120 ; biosynthesis ; genetics ; HIV Infections ; pathology ; Humans ; Liver ; pathology ; Male ; Middle Aged

OBJECTIVETo evaluate the technique and clinical outcomes of modified transperitoneal laparoscopic radical prostatectomy.

METHODSA total of 105 patients received the operation with age ranging from 51 to 73 years from January 2008 to June 2010. Mean level of serum prostate specific antigen was 13.6 µg/L and mean prostatic volume was 45 ml. Pathological studies of biopsy confirmed the prostate carcinoma with Gleason score 6-8. Radionuclide bone scan revealed no metastasis. Based on previously retroperitoneal radical prostatectomy, modified technique was applied involving surgical approach, bladder neck dissection and vesicourethral anastomosis.

RESULTSMean operative time was 93 min (65 - 150 min). Intraoperative blood loss was 115 ml (50 - 400 ml). No complication of bowl injury occurred. Positive surgical margin was present in 24 patients. Normal continence were seen in 64 patients after catheter removed. Recovery of incontinence within 3 months was seen in 33 patients and 3 to 12 months in 5 patients respectively. Three patients with incontinence were still in the follow-up.

CONCLUSIONSTransperitoneal laparoscopic radical prostatectomy provides large working space and clear anatomic exposure. Higher efficiency and lower complication rate are obtained through modified laparoscopic technique involving seminal vesicle isolation, bladder neck dissection and vesicourethral anastomosis.

Abdominal Cavity ; surgery ; Aged ; Humans ; Laparoscopy ; methods ; Male ; Middle Aged ; Prostatectomy ; methods ; Prostatic Neoplasms ; surgery ; Retrospective Studies

OBJECTIVETo investigate the effect of 5-Aza-2'-deoxycytidine (5-Aza-dC) on TIP30 gene expression and the relationship between TIP30 expression and the sensitivity to 5-fluouracil (5-Fu) in colorectal cancer cells.

METHODSThe methylation profile of TIP30 gene in HCT116 colorectal cancer cells was determined by methylation-specific PCR. The levels of TIP30 mRNA and protein were determined by RT-PCR and Western blot after the 5-Aza-dC treatment. MTT assay was used to detect the chemosensitivity of HCT116 cells to 5-Fu.

RESULTSTIP30 gene displayed complete DNA methylation in the HCT116 cells without 5-Aza-dC pretreatment. After the 5-Aza-dC treatment for 3 days, only demethylating PCR amplification product was detected and TIP30 gene showed DNA demethylation. With the prolongation of the time of removal of 5-Aza-dC treatment, methylated and demethylated PCR amplification products were observed and TIP30 gene displayed both DNA methylation and DNA demethylation in the colorectal cancer cells. At the day 10 after removal of 5-Aza-dC, methylating PCR amplification product appeared and TIP30 gene showed DNA methylation. No expressions of TIP30 mRNA and protein were detected in the HCT116 cells untreated with 5-Aza-dC. After the treatment of 5-Aza-dC for 3 d and then removed the 5-Aza-dC, the expressions of TIP30 mRNA and protein were increased obviously. With the prolonged time after 5-Aza-dC removal, the expressions of TIP30 mRNA and protein decreased and reached the lowest level on day 10. The IC50 values of 5-Fu were 41.62, 33.17 and 4.96 µg/ml in the HCT116 cells pretreated with 5-Aza-dC, d0 and d10 with the drug removal after drug treatment for 3 d, respectively.

CONCLUSIONSThe results of this study show that the expression of TIP30 gene may be associated with its DNA methylation status and may affect the sensitivity of colorectal cancer cells to 5-Fu.

Acetyltransferases ; genetics ; metabolism ; Antimetabolites, Antineoplastic ; pharmacology ; Azacitidine ; analogs & derivatives ; pharmacology ; Cell Proliferation ; drug effects ; CpG Islands ; genetics ; DNA Methylation ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Fluorouracil ; pharmacology ; Gene Expression Regulation, Neoplastic ; HCT116 Cells ; Humans ; Inhibitory Concentration 50 ; RNA, Messenger ; metabolism ; Transcription Factors ; genetics ; metabolism

We designed the specific primers and TaqMan probes targeting cytochrome b genes of mitochondrial DNA from bovine, goat and sheep. We used different fluorescents to label the probes. After optimization of reaction conditions, we set up a multiplex fluorescent real-time PCR method to detect bovine, goat and sheep derived materials, simultaneously. We finished the detection tests of 17 kinds of animal DNA and 200 DNA samples from different sources with the developed method and the National Standard GB/T 20190Y-2006 routine PCR method. The coincidence rate of these two methods was 100%. Without electrophoresis or restriction digestion, the developed method could reduce the test time to one third as routine PCR and identify three kinds of animal derived materials including bovine, goat and sheep in one reaction. The developed method was approximately 10 times more sensitive than routine PCR, and was applicable to identifications of bovine, goat and sheep derived materials in feed stuff, meat, milk, pelt and grease, etc. The study showed that the developed real-time PCR method is a rapid, sensitive and efficacious detection assay for bovine, goat and sheep derived materials in animal products.
Animal Feed ; analysis ; Animals ; Cattle ; Cytochromes b ; genetics ; DNA Primers ; DNA, Mitochondrial ; analysis ; genetics ; Food Contamination ; analysis ; Genes, Mitochondrial ; Goats ; Meat ; analysis ; Mitochondria, Muscle ; enzymology ; genetics ; Polymerase Chain Reaction ; methods ; Sheep

Objective To investigate the effect of atorvastatin preconditioning on cerebral myelin basic protein (MBP),glial fibrillary acidic protein (GFAP) and neurospecific enolase (NSE) in rat model of cerebral ischemia reperfusion.Methods A total of 30 male SD rats were randomly assigned to test group,model group and sham group.The rats of test group received atorvastatin 5 mg · kg-1 · d-1 by gastric gavage for 5 consecutive days before modling while the other two groups received the same volume of 0.9％ NaC1.Right middle cerebral artery occlusion (MCAO) ischemia-reperfusion model was established in both model group and test group,while sham group was only subjected to right middle cerebral artery separation and suture.The expressions of cerebral NSE,MBP and GFAP were measured with immunohistochemistry after 24 h reperfusion.Results The expressions of NSE,MBP and GFAP were 0.11 ±0.03,0.11 ±0.02,0.14 ±0.04 in model group,had significant differences with those in sham group,which were 0.18±0.02,0.11 ±0.00,0.19 ± 0.02 (P ＜ 0.05).The expressions of NSE and MBP in test group were 0.14 ± 0.02,0.14 ± 0.02,had significant differences with those of model group (P ＜0.05).The expression of GFAP in test group had no statistical significance with model group (P ＞ 0.05).Conclusion Atorvastatin preconditioning can alleviate cerebral ischemia reperfusion injury in rats with MCAO,probably through protecting oligodendrocytes and neurons.

Animals ; Chronic Disease ; Female ; Genetic Therapy ; Male ; Mesenchymal Stem Cell Transplantation ; Myocardial Infarction ; physiopathology ; therapy ; Swine ; Vascular Endothelial Growth Factor A ; genetics ; Ventricular Function, Left

OBJECTIVE: Exercise, as a common non-drug intervention, is one of several lifestyle choices known to reduce the risk of cancer. Mitochondrial division has been reported to play a key role in the occurrence and transformation of hepatocellular carcinoma (HCC). This study investigated whether exercise could regulate the occurrence and development of HCC through mitosis. METHODS: Bioinformatics technology was used to analyze the expression level of dynamin-related protein 1 (DRP1), a key protein of mitochondrial division. The effects of DRP1 and DRP1 inhibitor (mdivi-1) on the proliferation and migration of liver cancer cells BEL-7402 were observed using cell counting kit-8, plate colony formation, transwell cell migration, and scratch experiments. Enzyme-linked immunosorbent assay, Western blot and real-time polymerase chain reaction were used to detect the expression of DRP1 and its downstream phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway. A treadmill exercise intervention was tested in a nude mouse human liver cancer subcutaneous tumor model expressing different levels of DRP1. The size and weight of subcutaneous tumors in mice were detected before and after exercise. RESULTS: The expression of DRP1 in liver cancer tissues was significantly upregulated compared with normal liver tissues (P < 0.001). The proliferation rate and the migration of BEL-7402 cells in the DRP1 over-expression group were higher than that in the control group. The mdivi-1 group showed an inhibitory effect on the proliferation and migration of BEL-7402 cells at 50 μmol/L. Aerobic exercise was able to inhibit the expression of DRP1 and decrease the size and weight of subcutaneous tumors. Moreover, the expression of phosphorylated PI3K (p-PI3K) and phosphorylated AKT (p-AKT) decreased in the exercise group. However, exercise could not change p-PI3K and p-AKT levels after knocking down DRP1 or using mdivi-1 on subcutaneous tumor. CONCLUSION Aerobic exercise can suppress the development of tumors partially by regulating DRP1 through PI3K/AKT pathway.
Animals ; Apoptosis ; Carcinoma, Hepatocellular/therapy* ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Dynamins ; Liver Neoplasms/therapy* ; Mice ; Phosphatidylinositol 3-Kinase/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction

OBJECTIVE: Physical exercise, a common non-drug intervention, is an important strategy in cancer treatment, including hepatocellular carcinoma (HCC). However, the mechanism remains largely unknown. Due to the importance of hypoxia and cancer stemness in the development of HCC, the present study investigated whether the anti-HCC effect of physical exercise is related to its suppression on hypoxia and cancer stemness. METHODS: A physical exercise intervention of swimming (30 min/d, 5 d/week, for 4 weeks) was administered to BALB/c nude mice bearing subcutaneous human HCC tumor. The anti-HCC effect of swimming was assessed in vivo by tumor weight monitoring, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) detection of proliferating cell nuclear antigen (PCNA) and Ki67. The expression of stemness transcription factors, including Nanog homeobox (NANOG), octamer-binding transcription factor 4 (OCT-4), v-Myc avian myelocytomatosis viral oncogene homolog (C-MYC) and hypoxia-inducible factor-1α (HIF-1α), was detected using real-time reverse transcription polymerase chain reaction. A hypoxia probe was used to explore the intratumoral hypoxia status. Western blot was used to detect the expression of HIF-1α and proteins related to protein kinase B (Akt)/glycogen synthase kinase-3β (GSK-3β)/β-catenin signaling pathway. The IHC analysis of platelet endothelial cell adhesion molecule-1 (CD31), and the immunofluorescence co-location of CD31 and desmin were used to analyze tumor blood perfusion. SMMC-7721 cells were treated with nude mice serum. The inhibition effect on cancer stemness in vitro was detected using suspension sphere experiments and the expression of stemness transcription factors. The hypoxia status was inferred by measuring the protein and mRNA levels of HIF-1α. Further, the expression of proteins related to Akt/GSK-3β/β-catenin signaling pathway was detected. RESULTS: Swimming significantly reduced the body weight and tumor weight in nude mice bearing HCC tumor. HE staining and IHC results showed a lower necrotic area ratio as well as fewer PCNA or Ki67 positive cells in mice receiving the swimming intervention. Swimming potently alleviated the intratumoral hypoxia, attenuated the cancer stemness, and inhibited the Akt/GSK-3β/β-catenin signaling pathway. Additionally, the desmin⁺/CD31⁺ ratio, rather than the number of CD31⁺ vessels, was significantly increased in swimming-treated mice. In vitro experiments showed that treating cells with the serum from the swimming intervention mice significantly reduced the formation of SMMC-7721 cell suspension sphere, as well as the mRNA expression level of stemness transcription factors. Consistent with the in vivo results, HIF-1α and Akt/GSK-3β/β-catenin signaling pathway were also inhibited in cells treated with serum from swimming group. CONCLUSION Swimming alleviated hypoxia and attenuated cancer stemness in HCC, through suppression of the Akt/GSK-3β/β-catenin signaling pathway. The alleviation of intratumoral hypoxia was related to the increase in blood perfusion in the tumor. Please cite this article as: Xiao CL, Zhong ZP, Lü C, Guo BJ, Chen JJ, Zhao T, Yin ZF, Li B. Physical exercise suppresses hepatocellular carcinoma progression by alleviating hypoxia and attenuating cancer stemness through the Akt/GSK-3β/β-catenin pathway. J Integr Med. 2023; 21(2): 184-193.
Humans ; Animals ; Mice ; Carcinoma, Hepatocellular/drug therapy* ; Proto-Oncogene Proteins c-akt/metabolism* ; Proliferating Cell Nuclear Antigen/therapeutic use* ; Mice, Nude ; Glycogen Synthase Kinase 3 beta/genetics* ; beta Catenin/therapeutic use* ; Liver Neoplasms/drug therapy* ; Desmin/therapeutic use* ; Ki-67 Antigen ; Cell Line, Tumor ; Hypoxia ; RNA, Messenger/therapeutic use* ; Cell Proliferation