Effects of high-fat diet on inflammatory responses of ApoE-/- mice and migration of peripheral blood monocytes in atherosclerotic plaque / 西安交通大学学报(医学版)

【Objective】 To investigate the dynamic changes of local and systemic inflammatory responses after high-fat diet feeding of ApoE-/- mice so as to establish the method to assess the recruitment of PKH26-labeled monocytes into atherosclerotic plaque. 【Methods】 ApoE-/- mice that had received a high-fat diet (HFD) for 8 and 16 weeks were assessed for: ① atherosclerotic plaque burden (oil red O staining), ② aortic tissue inflammation (by RT-qPCR to detect aortic inflammatory factors and cells), ③ systemic inflammatory responses (by ELISA to measure plasma proinflammatory cytokine levels and flow cytometry to assess inflammatory cellular profile in the peripheral blood), and ④ recruitment of PKH26 labeled monocytes into atherosclerotic lesions (by confocal microscope). 【Results】 Compared to those in high-fat diet 8 weeks group, ApoE-/- mice in high-fat diet 16 weeks group had more serious atherosclerosis in the whole aorta and aortic root (P<0.01); significantly up-regulated aortic mRNA levels of inflammatory factors CCL2, TNF-α and IL-1β (P<0.01); an increased number of aortic local macrophages (P<0.001); significantly increased protein expression of plasma CCL2 and TNF-α (P<0.01); and the increased percentage of peripheral blood monocytes (P<0.05). Moreover, the number of PKH26 labeled monocytes that migrated to aortic lesions were significantly higher in high-fat diet 16 weeks group (P<0.01). 【Conclusion】 High-fat diet promotes the severity of systemic and local inflammatory responses in ApoE knockout mice, which contributes to the migration of peripheral blood monocytes to atherosclerotic plaque.

The feasibility of PKH26-marked human adipose derived mesenchymal stem cells and intraocular trace / 中华实验眼科杂志

Objective@#To evaluate the feasibility of PKH26 marked human adipose derived mesenchymal stem cells (hADSCs) in vitro and intraocular.@*Methods@#HADSCs were cultured in vitro and marked with PKH26.Eighteen C57BL/6J mice were divided into normal control group (6 mice), labeled cell group (9 mice) and unlabeled cell group (3 mice) by using sortition randomization method, labeled cells were injected intravitreally in C57BL/6J mice as labeled cell group, and the unlabeled cells were injected intravitreally in C57BL/6J mice as unlabeled cell group.After 1 month, retinal slab was checked to contrast the results of intraocular labeling.The retina was taken out for hematoxylin-eosin staining and electron microscopy to observe the toxicity.The use and care of the animals complied with Regulations for the Administration of Affair Concerning Experimental Animals by State Science and Technology Commission.@*Results@#In vitro, red fluorescent was found in the cytomembranes after hADSCs were labeled by PKH26.Retinal patch of labeled cell group showed red fluorescence of hADSCs were in front of the retina.Hematoxylin-eosin staining of retinal tissue and optic ganglion cells showed that cell degeneration and proliferation were not found in labeled cell group.The hADSCs in vivo and in vitro marked enhancement results showed that, no fluorescent was found in the unlabeled cell group, the color positive rate were both 0, while red fluorescence was found in the labeled cell group, the color positive rate were both 100%.@*Conclusions@#PKH26 can be used to mark and intraocular trace of hADSCs.Also, it has no morphology toxic reaction.

Stem cell-like properties of dormant cells in ovarian epithelial carcinoma transplantation tumors / 肿瘤

Objective: To examine the stem cell-like properties of dormant cells in ovarian epithelial carcinoma transplantation tumors. Methods: The PKH26-labeled human ovarian cancer SKOV3 cells were inoculated subcutaneously in nude mice. According to the fluorescence intensity of PKH2, the tumor cells were divided into 3 groups using FCM method: PKH26 cells with high fluorescence retention (PKH26hi cells), PKH26 cells with low fluorescence retention (PKH26low cells), and PKH26 cells with fluorescence quenched completely (PKH26neg cells). The cell cycle of PKH26hi, PKH26low and PKH26neg SKOV3 cells was detected by FCM method. The mRNAs and proteins of stem cell markers octamer-binding protein 3/4 (OCT3/4), CD1 33, CD1 77 and Nanog were examined by real-time fluorescence-based quantitative PCR and Western blotting. The colony forming ability of PKH26hi, PKH26low and PKH26neg SKOV3 cells in vitro was observed by plate colony formation assay, and the tumorigenic ability in vivo was observed in nude mice. Results: The percentage of G0/G1 cells in PKH26hi SKOV3 cells was higher than those in PKH26low and PKH26neg cells (both P < 0.05). The mRNA and protein expression levels of stem cell markers OCT3/4, CD1 33, CD1 1 7 and Nanog in PKH26hi were higher than those in PKH26low and PKH26neg SKOV3 cells (all P < 0.05). The abilities of in vitro colony formation of PKH26hi SKOV3 cells and their tumorigenesis in nude mice were significantly higher than those of PKH26low cells and PKH26neg SKOV3 cells (both P < 0.05). Conclusion: PKH26hi cells in ovarian cancer SKOV3 cell xenografts in nude mice have stem cell-like properties.

In vivo tracing of human amniotic mesenchymal stem cells labeled with PKH26 in rat intrauterine adhesions model / 生物工程学报

To observe the migration of human amniotic mesenchymal stem cells (hAMSCs) labeled with PKH26 in the endometrium of rats intrauterine adhesion. hAMSCs were isolated, identified and labeled with PKH26 to detect the biological characteristics of the cells. Rat intrauterine adhesion models were established using mechanical and infective method and PKH26-labeled hAMSCs were transplanted through the tail vein. The distribution of PKH26 labeled hAMSCs in the endometrium of rats were observed with the fluorescence confocal microscope. The results showed that PKH26 stain had no significant effect on cell activity, cycle, apoptosis and so on. PKH26-labeled positive cells were mainly distributed in injured endometrium of rats. It shows that the PKH26 labeling technique is a safe and effective method for tracing the human amniotic mesenchymal stem cells in the treatment of intrauterine adhesions.

PKH26 combined with Hoechst 33258 to trace the iRhom2 gene and its mutant proteins of Uncv mice in Vero cells / 中国比较医学杂志

Objective To determine the localization of iRhom2 and its mutant proteins of Uncv mice in Vero cells by PKH26 combined with Hoechst 33258 staining.Methods The cell membrane was stained with PKH26, and the nuclei were stained with Hoechst 33258 dye, and observed by laser scanning confocal microscopy.Results It was found that wild iRhom2 was distributed in the cytoplasm, and its iRhom2mut was present both in cytoplasm and cell nuclei.Conclusions The results of our study suggest that a deletion in N-terminal of iRhom2 affects its subcellular localization.

Preliminary Construction of Tissue Engineering Nucleus Pulposus Combining Silk Fibroin Porous Scaffold with Rabbit Nucleus Pulposus Cells / 天津医药

Objective To investigate the feasibility of construction of tissue engineering nucleus pulposus by com?bining the novel silk fibroin porous scaffold with PKH26 labeled rabbit nucleus pulposus cells. Methods Rabbit nucleus pulposus cells were isolated and cultured, then the passage 1 nucleus pulposus cells were stained with safranin O and typeⅡcollagen immunohistochemical staining. The isolated rabbit nucleus pulposus cells were labeled with PKH26. MTT assay was used for examining the proliferation of the nucleus pulposus cells before and after labeling. Labeled cells were inoculat?ed in the scaffold, cultured for 4 days and then the cell-scaffold complexes were implanted subcutaneously into nude mice. After 12 weeks of in vivo culture, the cell-scaffold complexes were detected by in vivo imaging technology, H&E staining, toluidine blue staining, safranin O staining and collagen typeⅡimmunohistochemical staining. Results Safranin O stain?ing and typeⅡcollagen immunohistochemical staining of the passage 1 nucleus pulposus cells were positive. The fluores?cence intensity of labeled cell was distributed, and the difference of OD value of nucleus pulposus cells was not statistically significant before and after labeling (P>0.05). The in vivo imaging technique showed a strong fluorescencea in porous scaf?fold. H&E staining of cell-scaffold complexes showed that the scaffolds were filled with a large number of nucleus pulposus cells and large amount of extracellular matrix. Toluidine blue staining, safranin O staining and typeⅡcollagen immunohisto?chemical staining were positive, and large amount of extracellular matrix was secreted around the cells. Conclusion The new silk fibroin porous scaffold with rabbit nucleus pulposus cells in vivo culture formed nucleus pulposus like tissue, which can be used for construction of tissue engineering nucleus.

Evaluation of Cartilage Engineering Using PKH26 and Molecular Light Imaging System / 天津医药

Objective To investigate the application of PKH26 and molecular light imaging system in cartilage en?gineering. Methods Canine chondrocyte was labeled by fluorescent dye PKH26 and seeded into the porous cartilage acel?lular matrix scaffold. The cells/scaffold constructs were cultured in vitro for 1 week. Then the constructs were implanted into the dorsal pocket of nude mice. We utilized a molecular light imaging system to macroscopically observe cells/scaffold con?structs in vivo with fluorescence at the 4th weeks, and compared with X-rays taken at the same position. The fluorescence im?ages were compared with the immunohistochemical and immunofluorescent results of cartilage-like tissue in vivo. Results Luminescent images were acquired at the 4th weeks, a red color enhanced overlay of the luminescent image over X-ray photo?graphic image demonstrated the location of the implants and the cell viability and cell growth on porous CACM scaffold in vivo were very well. Histological results show that the safranin O, anti-collagenⅡimmunohistochemistry and toluidine blue stain of cartilage-like tissue is positive. Immunofluorescence examination demonstrated chondrocytes in the constructs whitch is showen red fluorescence, and anti-collagenⅡimmunofluorescent staining was showen in green while the overlap?ping image is showen in yellow. Conclusion This study outlines an applicable non-destructive method to evaluate cell growth in tissue engineering constructs in vivo using PKH26 and molecular light imaging system.

Histolgical AND Immunohistochemical Study on the Effect of Stem Cell Therapy on Bleomycin Induced Pulmonary Fibrosis in Albino Rat

AIM OF WORK: To demonstrate the bleomycin induced histological changes in the lung and the possible protective and/or therapeutic effect of stem cell therapy. MATERIALS AND METHODS: Study was carried out on 36 adult male albino rats, classified into 4 groups: group I (control), group II (bleomycin treated group), group III (early stem cell treated group: immediately after bleomycin), group IV (late stem cell treated group: 7 days after bleomycin). Sections were taken at the 14th day of experiment. stained with Hematoxylin and Eosin, Masson's trichrome, immunohistochemichal stains for alpha-SMA & PCNA. Sections were examined by light & immunofluroscent microscopy. Area percent of collagen fibers, area percent & optical density of alpha-SMA immunopositive cells were measured as well as the number of H&E and PCNA stained pneumocytes type II was counted. RESULTS: Group II showed, thickening of septa, extravasation of blood, dividing pneumocytes type II cells with acinar formation, cellular infiltration, fibroblast cells, almost complete loss of normal lung architecture in certain fields, consolidation and replacement of the lung tissue with fibrous tissue in other fields. Restoring of lung tissue with significant decrease in mean area % of collagen fibers, alpha-SMA immunopositive cells were detected in group III. CONCLUSIONS: Early treatment with bone marrow derived mesenchymal stem cells (BMSCs) immediately after bleomycin administration showed a significant reduction in fibrotic changes, however the late treatment with BMSCs (7 days) after bleomycin administration showed non significant results.

An Experimental Study on Bovine Nucleus Pulposus Cells Labelled with PKH26 in Vitro / 天津医药

Objective To investigate the application of PKH26 fluorescent labeling on nucleus pulposus cells isolat-ed from bovine coccyx disc, and to provide nucleus pulposus tissue engineering with traceable nucleus pulposus cells by PKH26 fluorescence labelling. Methods Nucleus pulposus primary cells were isolated from the nucleus pulposus tissue de-tached from bovine coccyx disc by enzymatic digestion, and observed under the inverted microscope. Safranin O, toluidine blue and type Ⅱ collagen immunocytochemistry methods used to stain for passage one generation cells. Nucleus pulposus cells were labeled with PKH26 fluorescence in accordance with the instructions. The cell activity, fluorescence intensity at d0, d14 and d28 of culture, characteristics of proliferation and the expression of gene in labeled cells were assessed. Re-sults Isolated nucleus pulposus cells amounted to (1.56 ± 0.35) × 106/g. Under the inverted microscope, primary cells ad-hered at the 4 th day of culture, grew in groups, and covered the bottom of culture flask at the 13 th day. Both primary cells and the P1 generation cells were chondrocyte-like morphology. The staining of safranin O, toluidine blue and typeⅡcolla-gen immunocytochemistry for P1 generation of nucleus pulposus cells showed positive results. The cell activity before and af-ter PKH26 labeling showed more than 95%, and the fluorescence intensity at d0, d14 and d28 performed a decreasing trend, but still showed detect strong fluorescence at d28. There were no significant differences in proliferation and the expression of gene (collagen typeⅠandⅡ, aggrecan) before and after cell labeling (P>0.05). Conclusion As the seed cells of tissue en-gineering, nucleus pulposus cells isolated from bovine coccyx can reach a satisfactory number and maintain cartilage-like phenotype, and no changes shown in the biological characteristics after labeling. PKH26 labeled nucleus pulposus cells are suitable for the traceable cells in vivo study.

The Possible Role of Mesenchymal Stem Cells Therapy in the Repair of Experimentally Induced Colitis in Male Albino Rats

BACKGROUND AND OBJECTIVES: Colitis is inflammation of the colon which can be transmural or confined to the mucosa. Colitis may be acute or chronic. In case of serious intestinal discontinuity of epithelium, the regeneration capacity of local stem cells is not enough to complete tissue repair. Bone marrow mesenchymal stem cells (BM-MSCs) migrate into the gastrointestinal wall, where they may contribute to the repair progress. The present study aimed at evaluating the possible therapeutic effect of MSCs on induced colitis in albino rat. METHODS AND RESULTS: Twenty male albino rats were divided into 3 groups (control, colitis, MSCs), control group (4 rats), colitis group (8 rats) received once intra-rectal injection of 2 ml of 3% acetic acid. MSCs therapy group (8 rats) injected with MSCs 24 hours after colitis induction. In each group, rats were subdivided into subgroups (a & b). Subgroup (a) corresponds to rats sacrificed 3 days and subgroup (b) corresponds to rats sacrificed 10 days after colitis induction. Isolation and culture of MSCs from rat bone marrow were performed. Colon sections were examined using light and fluorescent microscopy. Colon specimens were subjected to histological, morphometric and statistical studies. In colitis group, ulceration, loss of surface columnar epithelium, disturbed crypts architecture with few goblet cells and huge lymphatic nodule piercing the muscularis mucosa were reported. In stem cell therapy group, MSCs stimulate colonic repair through differentiation into several cells and dampen the inflammation. CONCLUSIONS: MSCs represent future therapeutic hopes for intestinal injury and chronic intestinal inflammatory states.

Effect of Bone Marrow Derived Mesenchymal Stem Cells on Healing of Induced Full-Thickness Skin Wounds in Albino Rat

BACKGROUND AND OBJECTIVES: Mesenchymal stem cells have delivered new approaches to the management of wound healing in severe skin injuries. This work was planned to evaluate the effect of bone marrow-derived mesenchymal stem cells (BMSCs) on healing of induced full thickness skin wounds in albino rats using topical & systemic injections. METHODS AND RESULTS: Forty adult male albino rats were classified into 2 groups after induction of full thickness skin wound; untreated group and stem cell-treated group. The latter was further subdivided into topically and systemically treated ones. BMSCs were isolated & labeled by PKH26 before injection. Healing of wounds was evaluated grossly. Skin biopsies were obtained one & three weeks after wound induction. Sections were stained with Hematoxylin & Eosin, Masson's trichrome and immunohistochemichal stain for vascular endothelial growth factor (VEGF). Epidermal thicknesses and mean area percent of both collagen fibers & VEGF immunopositive cells were measured using image analyzer & results were subjected to statistical analysis. PKH26 fluorescent-labeled cells were found in the regenerated epidermis, hair follicles and dermis in BMSCs-treated groups. By the end of the third week, the wounds of BMSCs-treated groups showed full regeneration of epidermis, re-organization of collagen and decrease in VEGF immunopositive cells. Delayed wound healing was seen in 20% of systemically treated rats. Significant increase in the mean area percent of collagen fibers was detected in topically treated group. CONCLUSIONS: Both methods of BMSCs injection were effective in healing of full thickness skin wound but topical method was more effective.

Intrahepatic tracking of bone marrow-derived endothelial progenitor cells after transplanting into rats with liver fibrosis / 中华器官移植杂志

Objective To establish a method of PKH26 labeled bone marrow-derived endothelial progenitor cells (EPCs) into rats with liver fibrosis and observe cell immigration and differentiation in the liver after transplantation.Methods Bone marrow-derived EPCs were isolated and cultured from rats with liver fibrosis,and then labeled with PKH26 in vitro.Under the scanning confocal microscopy and flow cytometry,PKH26 fluorescent labeling rate and cell survival rate were measured.EPCs of PKH26 fluorescent labeling were transplanted into rats with liver fibrosis via the tail vein,and the migration situation was observed in the liver.Endothelial cell markers CD31 and von willebrand factor (vWF) were detected by using immunofluorescence.Results The PKH26-labeled EPCs appeared red fluorescence and the labeling rate was 96.65 ％.As compared with unlabeled cells,the labeled cells grew well,and had no significant changes in the growth curve.After transplantation into the liver of rats,the PKH26 labeled cells were mainly distributed in blood vessel endothelium along fibers and hepatic sinusoids in hepatic lobule.Endothelial cell-specific antigens such as CD31 and vWF could be detected along the vascular walls.Conclusion PKH26 could be used to label and track EPCs in the liver of rats with liver fibrosis in vitro.The PKH26-labeled cells may migrate to the surrounding of the hepatic vessels and differentiate into mature endothelial cells.