Preliminary experimental study on the prevention of all-trans retinoic acid induced cleft palate in mice by nicotinamide / 口腔疾病防治

Objective@# To explore the preventive effect of nicotinamide (NAM) on cleft palate induced by all-trans retinoic acid (RA), to provide research evidence for the prevention of cleft palate. @*Methods @#The mouse cleft palate model was induced by intragastric administration of 70 mg/kg all-trans retinoic acid at embryonic day 10.5 (E10.5) in the control group. The mouse cleft palate model was treated by caudal vein injection of 20 mg/kg NAM at E8.5 to E13.5 in the experimental group (1). The cleft palate model was treated by caudal vein injection of 40 mg/kg NAM at E8.5-E13.5 in the experimental group (2). The cleft palate of fetal rats was observed by laparotomy on E16.5 and statistically analyzed. Annexin V-FITC/PI double staining was used to detect the apoptosis of mouse embryonic palatal mesenchyme (MEPM) cells treated with RA 1 μmol/L (RA 1 group), NAM 200 μmol/L (NAM 200 group), and both NAM 200 μmol/L and RA 1 μmol/L (NAM 200+RA 1 group) for 24 hours by flow cytometry and the apoptosis rate in groups were compared. Culture without RA or NAM was used as a control. @*Results @# The cleft palate rate in the control group was 98%. The cleft palate rate in experimental group (1) was 87%. There was no significant difference between groups (P>0.05). The cleft palate rate in the experimental group (2) was 63%, compared with the control group, there was a significant difference (P<0.01). The cell apoptosis rate was 16.53%±2.89% in the CONTROL group. The cell apoptosis rate was 22.9%±1.85% in the RA 1 group, which was a significant increase compared with the CONTROL group (P<0.01). The apoptotic rate of the NAM 200 group was 9.23%±1.39%, which was a significant decrease compared with NA 1 group (P<0.01). The apoptosis rate of the NAM 200+RA 1 group was 14.9%±7.67%, which was a significant decrease compared with the RA 1 group (P<0.01).@*Conclusion@#NAM can prevent cleft palate. 40 mg/kg nicotinamide during pregnancy is an effective concentration for the prevention of RA-induced cleft palate. The mechanism by which NAM prevents cleft palate may be that NAM inhibits RA-induced apoptosis of MEPM cells.

Mechanism of lentivirus mediated flot1 gene expression on invasion and apoptosis of gastric cancer cells / 中国药学杂志

OBJECTIVE: To investigate inhibitory effect of FLOT1 gene expression on invasion and apoptosis of gastric cancer cells. METHODS: The designed FLOT1 siRNA lentiviral vector (si-FLOT1 group) was transfected into human gastric cancer MKN-45 cells, at the same time, the negative control lentivirus vector (negative group) was transfected, and the blank group was set up. Western blotting was used to detect the expression of FLOT1, E-cadherin, α-SMA, cleaved caspase 3, Bcl-2 and Bax proteins. After cells were transfected for 24, 48, 72 and 96 h, cell viability was detected by CCK-8 assay. After cells were transfected for 48 h, transwell chamber, flow cytometry and DCFH-DA assay were used to detect the invasiveness, apoptosis rate and ROS content, respectively. RESULTS: FLOT1 siRNA lentiviral vector inhibited significantly the expression of FLOT1 in MKN-45 cells, which was significantly different from the blank group (P<0.05). Compared with si-FLOT1 group and the blank group, the cell vitality decreased, the invasion ability decreased, the apoptosis rate increased, the expression of E-cadherin, cleaved caspase 3 and Bax protein increased, the expression of α-SMA and Bcl-2 protein decreased, and the content of ROS increased, and the difference was statistically significant (P<0.05). CONCLUSION: Down regulation of FLOT1 gene expression can reduce the invasiveness of gastric cancer cells by inhibiting EMT, and promote apoptosis by regulating the expression of apoptosis related proteins and increasing the ROS content of cells.

The relationship of MEOX2 and neurovascular dysfunction in Alzheimer's disease model / 药学学报

Present study investigated the role of mesenchyme homeobox 2 (MEOX2) gene in neurovascular dysfunction in Alzheimer's disease (AD) model rats by bilateral intracerebroventricular injection of Aβ1-42. One week after surgery, Morris water maze, immunohistochemistry, biochemical detection, Western blot and real-time PCR were used to detect the indexes. The animal studies were conducted in accordance with the Regulations of Experimental Animal Administration issued by the State Committee of Science and Technology of the People's Republic of China. Compared to the Sham-operated rats, Aβ1-42-operated rats showed obviously cognitive dysfunction, accompanied by increased Aβ, glial fibrillary acidic protein (GFAP), allograft inflammatory factor 1 (AIF1), endothelial nitric oxide synthase (eNOS) and decreased neuron specific enolase (NSE), synaptophysin (SYN), CD34, vascular endothelial growth factor (VEGF) expressions of brain. Aβ1-42-operated rats also increased the endothelin (ET) level and decreased nitric oxide (NO) level in brain tissue. Moreover, MEOX2 expression was decreased correlated with low density lipoprotein receptor-related protein 1 (LRP-1) decreasing and receptor for advanced glycation end products (RAGE) increasing in brain tissues of AD model rats. We found the correlation between MEOX2 gene expression and neurovascular dysfunction, in addition, the decreased MEOX2 may involve in increasing the accumulation of Aβ in brain by relating to the decreased LRP-1 and increased RAGE which is located in blood-brain barrier (BBB) in senescence-accelerated mice.

Progress in genetics of vesicoureteral reflux / 国际儿科学杂志

Vesicoureteral reflux,a most common congenital anomaly of the kidney and urinary tract,is associated with the malformation of ureterovesical junction. It does not cause any specific symptoms or signs un-less it is part of a syndrome or complicated by urinary tract infection. The exact cause is not clear,and genes or environmental factors may result in vesicoureteral reflux. The prevalence of siblings and offspring of reflux pa-tients are higher than normal control groups,so the genetic screening is necessary. This article will review the ge-netics of vesicoureteral reflux and possible interactions.

Inhibitory effect of all￣trans retinoic acid on osteogenic differentiation of mouse embryonic palate mesenchymal cells and its possible mechanism / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect and related mechanism of all￣trans retinoic acid (atRA) exposure on osteogenic differentiation of mouse embryonic palate masenchymal cells MEPM. METHODS MEPM were cultured in osteogenic medium (OM) with atRA 0.1 and 1.0 μmol??L-1 for 1, 3,5, 7 and 9 d. MTT assay was performed to measure the cell viability. The alkaline phosphatase (ALP) activity was measured by chemical colorimetry. The cells were stained using the Von￣Kossa technique to detect the formation of mineralization nodules after 21 d of culture. RT￣PCR was performed to determine expression Runx2, osteopontin, bone morphogenetic protein receptor ( Bmpr) 1b, Bmpr2 and Smad5 mRNA. RESULTS The result of MTT on 9 d showed that, compared with normal control group, the cell viability of OM, OM+atRA 0.1 and 1.0 μmol??L-1 groups decreased significantly(P<0.01). Compared with normal control group, ALP activity of OM group increased significantly(P<0.05), while the ALP activity of OM+atRA 0.1 and 1.0 μmol??L-1 groups was lower than OM group(P<0.05). On 21 d, the Von￣Kossa stai￣ning results showed that the percentage of mineralization nodules formation of OM+atRA 1.0 μmol??L-1 group was (3.65±1.24)%, which was significantly lower than that of OM group(10.33±2.29)%(P<0. 05). On 9 d, the relative Run expression of OM group was the highest one in the four groups, while at￣RA 1.0 μmol??L-1 treatment negatively regulated 20% in comparsion with OM group(P<0.05). Compared with normal control group, the mRNA expression of osteopontin of OM, OM+atRA 0.1 and 1.0 μmol??L-1 groups increased significantly(P<0.05); BDNF mRNA expression of OM group was 2.6￣fold to normal control group, while that of OM+atRA 1.0 μmol??L-1 group was 33% to OM group(P<0.05) . The level of Smad5 mRNA of OM+atRA 1.0 μmol??L-1 group was significantly lower than that of OM group(P<0.05). CONCLUSION atRA Might inhibit osteogenic differentiation of MEPM by down￣regulated the expression of Bmpr1b.

Molecular regulation of kidney development / 대한해부학회지

Genetically engineered mice have provided much information about gene function in the field of developmental biology. Recently, conditional gene targeting using the Cre/loxP system has been developed to control the cell type and timing of the target gene expression. The increase in number of kidney-specific Cre mice allows for the analysis of phenotypes that cannot be addressed by conventional gene targeting. The mammalian kidney is a vital organ that plays a critical homeostatic role in the regulation of body fluid composition and excretion of waste products. The interactions between epithelial and mesenchymal cells are very critical events in the field of developmental biology, especially renal development. Kidney development is a complex process, requiring inductive interactions between epithelial and mesenchymal cells that eventually lead to the growth and differentiation of multiple highly specialized stromal, vascular, and epithelial cell types. Through the use of genetically engineered mouse models, the molecular bases for many of the events in the developing kidney have been identified. Defective morphogenesis may result in clinical phenotypes that range from complete renal agenesis to diseases such as hypertension that exist in the setting of grossly normal kidneys. In this review, we focus on the growth and transcription factors that define kidney progenitor cell populations, initiate ureteric bud branching, induce nephron formation within the metanephric mesenchyme, and differentiate stromal and vascular progenitors in the metanephric mesenchyme.

Expression of Robo2 gene during the renal development in mice / 解放军医学杂志

Objective To observe the expression of Robo2 gene, and explore its role during the renal development of mice. Methods Real-time quantitative RT-PCR was used to semi-quantitatively measure the expression level of Robo2 mRNA in the developing murine kidney at fetal age of 12.5, 13.5, 14.5, 15.5, 16.5 and 17.5 days, and also 1 day, 1 week, 5 weeks after birth. Immunofluorescence staining was used to examine the expression location of Robo2 protein at different stages of embryonic and postnatal kidney. Results Real-time quantitative RT-PCR analysis revealed that Robo2 was highly expressed in embryonic kidney at fetal age of 12.5, 13.5 and 14.5 days, while the expression level declined quickly thereafter and maintained at very low level after birth. Immunofluorescence staining showed that the expression of Robo2 protein could be primarily detected in metanephric mesenchyme of the developing kidney, but not in the ureteric bud. With the development of embryonic kidney, Robo2 protein was expressed in cell membrane of metanephric mesenchyme, condensed cap mesenchyme surrounding the tip of the ureteric bud, comma-shaped body, S-shaped body and renal capsule, finally expressed in the podocytes. Besides, Robo2 protein was also weakly expressed in part of the proximal tubular epithelial cells. Absence of Robo2 gene resulted in abnormal development of nephron, and broadening of some renal tubules and collecting ducts. Conclusion Robo2 plays an important role in the nephron development in mice by regulating the interaction of metanephric mesenchyme and ureteric bud.

An experimental study of folic acid rivaling methylenetetrahydrofolate reductase (MTHFR) gene / 实用口腔医学杂志

Objective: To further understand the role of folic acid supplements rivaling MTHFR gene silencing in pathogenesis of NCLP, RNA interference (RNAi) was applied to knock down MTHFR in mouse embryonic palatal mesenchymal (EPM) cells. Methods: MTHFR ShRNA expression vector were transfected into the primary cultured EPM cells. MTT was used to observe cell proliferation after MTHFR gene silencing. FCM was used to observe cell cycle after MTHFR gene silencing. Results: The results showed the cells proliferation had an inequality amelioration after using folic acid supplements in MEPM cells with MTHFR gene silencing. Using folic acid supplements rivaled the effect of MTHFR gene silencing had a dose-dependent manner. Using 20 μg/ml folic acid supplements could improve the cell proliferation to achieve normal level of cell proliferation. Conclusion: MTHFR gene is an important candidate gene of NCL/P. Using folic acid supplements could prevent teratogenic MTHFR gene silencing for embryonic palate development.

Intranodal Palisaded Myofibroblastoma with Desmin Expression : A Brief Case Report

Intranodal palisaded myofibroblastoma is a rare benign mesenchymal neoplasm of the lymph node. It is characterized by intranodal spindle cell proliferation along with amianthoid fibers and prominent hemorrhage. It has been rarely reported in South Korea. We report here on a case of palisaded myofibroblastoma that arose in the left inguinal lymph node. The tumor mass was well demarcated, and it was composed of a proliferation of benign-looking spindle cells. It showed focal hemorrhage and a fibrous pseudocapsule. The tumor cells displayed little pleomorphism, no mitotic count, and characteristic palisading nuclei and amianthoid fibers. The tumor cells were positive for smooth muscle actin, vimentin, and also for desmin, but they were negative for S-100 protein, supporting the diagnosis of myofibroblastoma.

Solitary Fibrous Tumor as Misdiagnosed as Bladder Cancer / 대한비뇨기과학회지

Solitary fibrous tumor(SFT) was previously named localized fibrous mesothelioma, and this is a rare mesenchymal neoplasm that usually shows benign behavior. It is the most commonly recognized tumor of the pleura. These tumors have been recently reported to be found in unexpected locations. To the best of our knowledge, there have been only 2 reports of SFT arising from the urinary bladder in Koreans. We review this third case of SFT that was misdiagnosed as bladder cancer. This tumor was removed from a 40-year-old man who had a history of lower urinary tract symptoms.

Study of hTERT activating telomerase in human bone marrow mesenchyme stem cells / 中国矫形外科杂志

[Objective]To explicate whether the telomerase activity is regulated by human telomerase reverse transcriptase (hTERT) in human bone marrow mesenchyme stem cells(hBMSC).[Method]hBMSC were cultured and transfected with eukaryotic expressing plasmid pCIneo-hTERT encoding hTERT. After selection with G418 to stabilize the transfection,expression of hTERT mRNA was detected with TR-PCR, detecting the expression of hTERT protein was detected with Western Bolt, and the telomerase activity in untransfected and transfected cells were detected by RT-PCR.[Result]The hMSCs grew well after transfecting plasmid pCIneo-hTERT.The cells began to suspend and die after the day of the G418 selection. At the tenth day,all the untransfected cells were dead, but the transfected cells began to clone proliferation. So the density of G418 subdued to 100 ?g/ml for maintaining selecting, at the twentieth day,there were obvious anti-G418 cell clones. After stable transfection, hTERT was expressed at mRNA and protein level in these anti-418 cell clones, and meanwhile telomerase activity was positive and obviously raise up in these transfected cells.[Conclusion]In human' bone marrow mesenchyme cells,telomerase could be activated by exogenous hTERT. This is a foundation to establish immortalized human bone marrow mesenchyme stem cell line.

Solitary Fibrous Tumor of Bladder / 대한비뇨기과학회지

A solitary fibrous tumor (SFT) is now commonly accepted to derive from mesenchymal cells differentiating toward fibroblast or myofibroblast. Although the extrapleural manifestations of SFT have been documented in almost all sites, an SFT arising in the genitourinary tract is extremely rare, with less than 10 cases having been reported. The histopathological criteria between a benign and a malignant SFT are obscure, and their biological behaviors remain controversial. The choice of treatment of an SFT remains to be clarifies. Herein, a case of a bladder SFT, well encased within the submucosa and bladder muscle, is reported. The SFT of bladder was completely excised, and there was no evidence of recurrence after 15 months of follow-up.

Transdifferentiation of Cultured Bovine Lens Epithelial Cells into Myofibroblast-like Cells by Serum Modulation

An after-cataract is caused by the proliferation of residual cells over the equator of the lens. These cells subsequently migrate to the posterior lens capsule, where they undergo aberrant differentiation into fiber-like cells or transdifferentiation into fibroblast-like cells. To study the precise molecular mechanisms of transdifferentiation, an attempt was made to establish an in vitro system, in which the lens epithelial cells (LECs) of the pre-equatorial zone could be transdifferentiated into fibroblast-like cells. The required conditions for culturing the LECs were identified as consisting of four phases; intact bovine explants, explant-cultured, serum-modulated and additionally modulated LECs. The LECs of each phase were compared by examining changes in the expression of the epithelial-mesenchymal transition (EMT) -related genes and changes in cellular morphology and adhesion. The explants that were cultured in a medium containing 10% fetal bovine serum (FBS) for 2 weeks, showed changes in the expression of the EMT-related genes, although the other explant-cultured cells maintained an epithelial morphology. To introduce a transition into mesenchymal cells, the explant cultures were subcultured in a medium containing 20% FBS for six passages. These cells displayed an elongated morphology and were able to grow and migrate in a similar way to fibroblast cells. The expression of the EMT-related genes, such as, extracellular matrix proteins and integrins, was altered. This was similar to the alteration of the 3-dimensional collagen gels model previously reported. During a further process of EMT by additional serum modulation, the inhibitory effect of disintegrin on cell adhesion was gradually decreased, integrin expression was differentially regulated and alpha-smooth muscle actin was post-translationally modified from the point of passage number six. Overall, it can be concluded that terminal transdifferentiation accompanies changes in the cytoskeletal proteins and cell surface molecules. These are modulated in systematic patterns of post-transcriptional and post-translational regulation and patterns of gene regulation, by the synergic effects of several transforming factors contained in serum. Therefore, posterior capsular opacification may also be accompanied by this molecular mechanism.

A Case of Subcutaneous Folliculosebaceous Cystic Hamartoma / 대한피부과학회지

Folliculosebaceous cystic hamartoma(FSCH) is a rare cutaneous hamartoma of follicular, sebaceous, and mesenchymal elements. The tumor usually has sessile or pedunculated papule or nodule and occurs frequently in the center of face and sometimes on the scalp, ear, and trunk. We report a case of FSCH, which a 36-year-old woman presented as a subcutaneous nodule on the occipital area of scalp. Histologic examination of the nodule showed a central large cystic structure connected with numerous sebaceous lobules, and stroma consisted of delicate fibrillary bundles of collagen in concert with dilated capillaries and venules, as well as with adipocytes.