Objective:To investigate the roles of secretory phosphoprotein 1(SPP1)in the progression of colorectal cancer(CRC)and the underlying mechanism.Methods:Gene Expression Profiling Interactive Analysis(GEPIA)database was used to obtain the expression of SPP1 gene in CRC.Immunohistochemistry analysis and Western blotting were used to detect the expression of SPP1 in distal normal colorectal tissues,adjacent tissues,CRC tissues,normal colorectal cell lines and CRC cell lines.The cell viability,colony formation,migration and invasion of CRC cells as well as the activation of AKT/glycogen synthase kinase 3β(GSK3β)signaling pathway and the expression of epithelial-mesenchymal transition(EMT)-related proteins in HT-29 cells and HCT-116 cells were detected by CCK-8 assay,colony formation assay,trranswell assay and Western blotting after SPP1 knockdown in vitro through lentiviral infection carrying shRNA against SPP1 gene.Tumor formation assay was used to detect the effect of SPP1 knockdown on the growth and lung metastasis of transplanted HT-29 tumor in vivo.Results:SPP1 expression was significantly increased in CRC tissues and cell lines(P<0.001)and was associated with poor prognosis of CRC patients according to GEPIA database analysis.The expression of SPP1 protein was significantly upregulated in CRC tissues and cells(P<0.001).After knockdown of SPP1 expression,the cell viability,colony formation,migration and invasion of CRC cells were significantly decreased(P<0.001),the expression of phosphorylated AKT(phospho-AKT,p-AKT),phosphorylated GSK3β(phospho-GSK3β,p-GSK3β),Snail and Vementin were significantly decreased(P<0.001),while E-cadherin expression was significantly increased(P<0.001).Knockdown of SPP1 expression inhibited the growth and lung metastasis of HT-29 cell tumor xenografts in mice.Conclusion:SPP1 knockdown can inhibit the proliferation,migration and invasion of CRC cells,which may be related to the reduction of AKT/GSK3β signaling activity.

Objective:To investigate the roles of secretory phosphoprotein 1(SPP1)in the progression of colorectal cancer(CRC)and the underlying mechanism.Methods:Gene Expression Profiling Interactive Analysis(GEPIA)database was used to obtain the expression of SPP1 gene in CRC.Immunohistochemistry analysis and Western blotting were used to detect the expression of SPP1 in distal normal colorectal tissues,adjacent tissues,CRC tissues,normal colorectal cell lines and CRC cell lines.The cell viability,colony formation,migration and invasion of CRC cells as well as the activation of AKT/glycogen synthase kinase 3β(GSK3β)signaling pathway and the expression of epithelial-mesenchymal transition(EMT)-related proteins in HT-29 cells and HCT-116 cells were detected by CCK-8 assay,colony formation assay,trranswell assay and Western blotting after SPP1 knockdown in vitro through lentiviral infection carrying shRNA against SPP1 gene.Tumor formation assay was used to detect the effect of SPP1 knockdown on the growth and lung metastasis of transplanted HT-29 tumor in vivo.Results:SPP1 expression was significantly increased in CRC tissues and cell lines(P<0.001)and was associated with poor prognosis of CRC patients according to GEPIA database analysis.The expression of SPP1 protein was significantly upregulated in CRC tissues and cells(P<0.001).After knockdown of SPP1 expression,the cell viability,colony formation,migration and invasion of CRC cells were significantly decreased(P<0.001),the expression of phosphorylated AKT(phospho-AKT,p-AKT),phosphorylated GSK3β(phospho-GSK3β,p-GSK3β),Snail and Vementin were significantly decreased(P<0.001),while E-cadherin expression was significantly increased(P<0.001).Knockdown of SPP1 expression inhibited the growth and lung metastasis of HT-29 cell tumor xenografts in mice.Conclusion:SPP1 knockdown can inhibit the proliferation,migration and invasion of CRC cells,which may be related to the reduction of AKT/GSK3β signaling activity.

In order to reduce osmotic damage and chemical toxicity of cryoprotectants (CPA) to oocytes during unloading process, the microfluidic chip was used to remove CPA from porcine MⅡ oocytes in this study. Firstly, the effects of unloading time, composition and concentration of diluting solutions of microfluidic method on survival rate and developmental capacity of oocytes were studied, then microfluidic method was compared with traditional one-step and two-step CPA unloading protocols. The results showed that when the total time is 8 minutes, the survival rate and morula rate of oocytes treated with microfluidic method could achieve 95.99% ± 4.64% and 74.17% ± 1.18%, respectively, which were not significantly different from fresh control group (98.53% ± 2.94%; 78.22% ± 1.34%). In addition, 1 mol/L sucrose diluting solutions were more beneficial than other solutions, and it was also showed that microfluidic method achieved better survival, cleavage rate of oocytes than traditional methods. Microfluidic CPA removal protocol can reduce the damage to oocytes during unloading process, and may further improve the cryopreservation effect of oocytes.

The purpose of this study was to study the effect of three different transfection reagents (Lipofectamine™ LTX & PLUS™, Lipofectamine 2000 and Nano-PAMAM-D) and three different testicular injection methods (rete testicular injection, seminiferous tubules injection and testicular interstitial injection) on the efficiency of production transgenic mice. After the mixtures of plasmid DNA (pEFP-C1) and transfection reagent were injected with different testicular injection methods, the sperm density, vitality, positive sperm rates and PCR positive transgenic mice rate were examined 30 days after injection. The results showed that the damage degree from slight to serious of three transfection reagents was Lipofectamine™ LTX & PLUS™, Lipofectamine 2000, and PAMAM-D. The sperm positive rates with green fluorescence of these three groups were 35.65%±0.69%, 12.86%±0.35% and 10.04%±0.20%, respectively. The PCR positive rates of transgenic newborn mice were 29.17%, 13.70% and 5.88%, respectively. Among the groups of different testicular injection methods, the damage degree from slight to serious was rete testicular injection, seminiferous tubules injection, and testicular interstitial injection, whereas the sperm positive rates with green fluorescence were 35.13%, 15.13%, and 0%, respectively. The PCR positive rates of transgenic newborn mice among different testicular injection groups were 33.3%, 12.5%, and 0.0%. The combination of rete testicular injection and Lipofectamine™ LTX & PLUS™ had the lowest toxicity and highest transgenic efficiency in the production of transgenic mice.
Animals ; Humans ; Indicators and Reagents ; chemistry ; Injections ; methods ; Lipids ; chemistry ; Male ; Mice ; Mice, Transgenic ; Plasmids ; Polymerase Chain Reaction ; Seminiferous Tubules ; Spermatozoa ; Testis ; Transfection

Nano-cryopreservation may become a new way in the next generation of cryopreservation technology. However, research using nanoparticles in oocytes vitrification has not been reported in the literature. In this study, HA nanoparticles with different diameters were added into cryoprotectant and M II-stage porcine oocytes were vitrified by Cryotop. The results showed that nanoparticles improved the survival rate of cryopreserved M II-stage porcine oocytes, but the difference between nanoparticles with different diameters of was not significant. In order to study the mechanism of nano-cryopreservation, the cooling rate of cryoprotectant was measured by ultra-fast temperature measurement system and the melting enthalpy of cryoprotectant was measured by differential scanning calorimeter (DSC). The results showed that the adding of nanoparitcles could not increase the cooling rate of cryoprotectant, but could decreases the amount of ice crystals during freezing and warming. Therefore, the mechanical injury within and outside cells might be effectively reduced.
Animals ; Cell Survival ; physiology ; Cryopreservation ; methods ; veterinary ; Cryoprotective Agents ; pharmacology ; Durapatite ; pharmacology ; Female ; Fertilization in Vitro ; methods ; veterinary ; Metaphase ; Nanoparticles ; Oocytes ; cytology ; Swine ; Vitrification

After freeze-drying, the ultrastructure of boar sperms was observed by optical and electron microscopy. The in vitro development ability of the sperm was also examined with intracytoplasmic sperm injection (ICSI). The rate of male pronuclear formation was (68.52%), for cleavage (59.17%) and for blastocyst formation (19.16%) of the trehalose group (0.2 mol/L), significantly higher than those of the 50 mmol/L EDTA group (64.59%, 56.26% and 15.62%) and the control group (35.36%, 52.33% and 8.60%) (P < 0.05). After storage for 60, 120 and 180 d at 4 degrees C, no significant difference in the in vitro development was observed (P > 0.05). The male pronuclear, cleavage and blastocyst formation after ICSI with freeze-dried spermatozoa incubated for 1 h was superior than those incubated for 2 h (P < 0.05). No significant differences in the structures after stored at 4 degrees C or -20 degrees C (P > 0.05) were observed between the trehalose group and EDTA group. The percent of B grade freeze-dried boar spermatozoa in the trehalose group was higher than that of the EDTA group (P < 0.05). Based on the ultrastructure observation, main cryogenic damage in freeze-dried boar spermatozoa was swelling, damage or rupture in the sperm acrosome, neck and tail.
Animals ; Freeze Drying ; Male ; Semen Preservation ; methods ; veterinary ; Sperm Injections, Intracytoplasmic ; veterinary ; Spermatozoa ; Swine ; Trehalose ; pharmacology

The purpose was to optimize the vitrification for porcine embryos cryopreservation. Blastocyst/Morula (5-6th day-embryos) were collected from superovulated Bama mini-pigs (sows/gilts). We compared different cryopreservation methods, cryopreservation tools, thining of zona pellucida (ZP) and recipient breeds on the efficiency of porcine embryo cryopreservation. The results showed that: in embryo survival rate and blastocyst cell number, there were no significant differences between cryopreservation method I [embryos were vitrified by two step method with open pulled straw (OPS) and glass micropipette (GMP) in solution 1 (TCM199 + 20% FBS + 10% EG + 10% DMSO) for 3 min, and solution 2 (TCM199 + 20% FBS + 20% EG + 20% DMSO + 0.4 mol/L SUC) for 1 min, stored in liquid nitrogen] and method II[Blastocysts were cultured for 25 min in NCSU23 + 7.5 microg/mL cytochalasin B, centrifuged at approximately 13 000 xg for 12-13 min, and recovered back into pNCSU23. They were then equilibrated for 5 min in 2 mol/L ethylene glycol in pNCSU23, washed quickly in the vitrification medium, 8 mol/L ethylene glycol, 7% polyvinylpyrrolidone (PVP) in pNCSU23, loaded into OPS/GMP, and plunged into liquid nitrogen]. GMP vitrification method was more suitable and efficient than OPS method (P < 0.05) in embryo survival rate (83.8% vs 77.6%) and blastocyst cell number (53.1 vs 47.5) after thawing. Thining of ZP did not increase the survival rate, but significantly improved blastocyst cell number in the survival blastcysts (60.1 and 46, P < 0.01). Local pig breeds (Fengjing sows) were more suitable as recipients for embryo transfer of vitrified/warmed blastcysts, which can improve pregnant rate and embryo efficiency.
Animals ; Blastomeres ; cytology ; Cryopreservation ; methods ; veterinary ; Embryo Transfer ; veterinary ; Embryo, Mammalian ; Swine ; Swine, Miniature ; Vitrification ; Zona Pellucida ; physiology