Osteogenic Response of MC3T3-E1 and Raw264.7 in the 3DEncapsulated Co-Culture Environment

BACKGROUND@#Three-dimensional (3D) in vitro cultures recapitulate the physiological microenvironment and exhibit high concordance with in vivo conditions. Improving co-culture models with different kind of cell types cultured on a 3D scaffold can closely mimic the in vivo environment. In this study, we examined the osteogenic response of pre-osteoblast MC3T3-E1 cells and Raw264.7 mouse monocytes in a 3D-encapsulated co-culture environment composed of the Cellrix®3D culture system, which provides a physiologically relevant environment. @*METHODS@#The Cellrix® 3D Bio-Gel scaffolds were used to individually culture or co-culture two type cells in 3D microenvironment. Under 3D culture conditions, osteoblastic behavior was evaluated with an ALP assay and staining. ACP assay and TRAP staining were used as osteoclastic behavior indicator. @*RESULTS@#Treatment with osteoblastic induction factors (?3F) and RANKL had on positively effect on alkaline phosphatase activity but significantly inhibited to acid phosphatase activity during osteoclastic differentiation in 3D coculture. Interestingly, alkaline phosphatase activity or acid phosphatase activity in 3D co-culture was stimulated with opposite differentiation factors at an early stage of differentiation. We guess that these effects may be related to RANK– RANKL signaling, which is important in osteoblast regulation of osteoclasts. @*CONCLUSION@#In this study, the osteogenic response of 3D encapsulated pre-osteoblast MC3T3-E1 cells and mouse monocyte Raw264.7 cells was successfully demonstrated. Our 3D culture conditions will be able to provide a foundation for developing a high-throughput in vitro bone model to study the effects of various drugs and other agents on molecular pathways.

Osteogenic Response of MC3T3-E1 and Raw264.7 in the 3DEncapsulated Co-Culture Environment

BACKGROUND@#Three-dimensional (3D) in vitro cultures recapitulate the physiological microenvironment and exhibit high concordance with in vivo conditions. Improving co-culture models with different kind of cell types cultured on a 3D scaffold can closely mimic the in vivo environment. In this study, we examined the osteogenic response of pre-osteoblast MC3T3-E1 cells and Raw264.7 mouse monocytes in a 3D-encapsulated co-culture environment composed of the Cellrix®3D culture system, which provides a physiologically relevant environment. @*METHODS@#The Cellrix® 3D Bio-Gel scaffolds were used to individually culture or co-culture two type cells in 3D microenvironment. Under 3D culture conditions, osteoblastic behavior was evaluated with an ALP assay and staining. ACP assay and TRAP staining were used as osteoclastic behavior indicator. @*RESULTS@#Treatment with osteoblastic induction factors (?3F) and RANKL had on positively effect on alkaline phosphatase activity but significantly inhibited to acid phosphatase activity during osteoclastic differentiation in 3D coculture. Interestingly, alkaline phosphatase activity or acid phosphatase activity in 3D co-culture was stimulated with opposite differentiation factors at an early stage of differentiation. We guess that these effects may be related to RANK– RANKL signaling, which is important in osteoblast regulation of osteoclasts. @*CONCLUSION@#In this study, the osteogenic response of 3D encapsulated pre-osteoblast MC3T3-E1 cells and mouse monocyte Raw264.7 cells was successfully demonstrated. Our 3D culture conditions will be able to provide a foundation for developing a high-throughput in vitro bone model to study the effects of various drugs and other agents on molecular pathways.

Expression and significance of PHH3 in pancreatic neuroendocrine neoplasms / 中华胰腺病杂志

Objective To investigate the expression of phospho-histone H3 (PHH3) protein in pancreatic neuroendocrine neoplasms (PNENs) and explore its potential value for pathological grading of PNENs.Methods Clinical and pathological data of 283 patients with PNENs treated in Changhai Hospital from December 2000 to May 2016 were retrospectively analyzed.PNENs tissue chip was prepared,and immunohistochemistry was used to examine the expression of PHH3 and Ki67.The receiver operating characteristic curve for PHH3 was drawn and the area under the curve(AUC) was calculated to determine the cutoff value for PNENs grading.Ki67 was used for PNENs grading according to domestic and intemational criteria for PNENs classification.The relationship of PHH3 and Ki67 classification with clinical pathological features and prognosis of PNENs as well as the correlation between the two classification methods were analyzed.Results Of 283 patients,132 were male and 151 were female,aged from 16 to 78 years old.The average age was (50 ± 1) years old.There were 44 cases with functional PNENs and 239 with non-functional PNENs.The diameter of tumors ranged from 1.1 cm to 17 cm and the average diameter was (4.1 ± 1.2)cm.According to the Ki67 standard,there were 127,116,33 and 7 cases of Grade G1,G2,pancreatic neuroendocrine tumor(PNET) G3 and pancreatic neuroendocrine cancer(PNEC) G3.According to the PHH3 standard,there were 118,119,3 8 and 8 cases of Grade G1,G2,PNET G3 and PNEC G3.There was a positive correlation between the two grading criteria (r =0.941,P ＜0.001).PHH3 expression and PNENs grading were not correlated with age,gender,tumor functional or not and tumor locations (all P ＞ 0.05),but were both positively correlated with tumor size,histological grade,lymph node metastasis,TNM stage and prognosis (all P ＜0.05).The average time of observing PHH3 and Ki67 staining per case in the immunohistochemistry of tissue chip was 9 min and 45 min,respectively.Conclusions Detection of PHH3 expression in PNENs can be used for the pathological diagnosis,grading and prognostic evaluation,which was more accurate and convenient than Ki67 criteria.

Preliminary analysis of bitter substances in spica of Prunella vulgaris / 中国中药杂志

Volatile oil components and the contents and types of amino acid in spica of Prunella vulgaris were analysed by GC-MS and amino acid analyzer. Esters, fatty acids, aromatic hydrocarbon, ketone and several alcohol compounds were identified by mass spectrum comparison. In these ingredients, beta-ionone smelled aroma of cedar, raspberry, nerolidol showed weak sweet soft orange blossom flavor, neroli tasted sweet and fresh, nerolidol tasted sweet with light aroma of wood, hexadecanal showed a weak aroma of flowers and wax, alpha-sinensal had rich and fresh sweet orange flavor. To some extent, these types of aromatic substances can affect the taste of herbal tea or decoction made of Spica Prunellae. Among amino acids detected, natural amino acids accounted for a larger proportion, and those natural amino acids showed bitterness, slight bitterness, sourness (freshness), sweetness, slight sweetness, sourness (slight freshness). The results indicated that bitter and slightly bitter amino acids have the greatest impacts on the sense of Spica Prunellae.

Inhibitory effects of soluble endoglin on invasive ability of cultured cytotrophoblasts of first trimester of pregnancy / 生理学报

The present study aims to investigate the effects of soluble endoglin (sEng) on invasive ability of cultured cytotrophoblasts of first trimester of pregnancy. Cytotrophoblasts of normal 6 to 8-week pregnancy were cultured by trypsin digestion method, and were incubated with cell culture medium without (control group) and with 10 μg/L sEng (sEng group), respectively for 24 h. The invasive ability was determined by transwell invasion assay, and expressions of MMP-2, MMP-9 mRNA and protein were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot, respectively. The results showed that the invasive ability of cytotrophoblasts in sEng group was lower than that in control group (P < 0.05). Compared with control group, the expressions of MMP-2 and MMP-9 mRNA and protein of cytotrophoblasts were significantly lower (P < 0.05). In conclusion, sEng may participate in the genesis of preeclampsia by affecting the invasive ability of cytotrophoblasts through regulation of the expression of MMP-2 and MMP-9.

Overexpression of connexin 45 in rat mesenchymal stem cells improves the function as cardiac biological pacemakers / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Extensive research toward creating a biological pacemaker by enhancement of inward depolarizing current has been performed. However, studies have mainly focused on inducing spontaneous activity and have not adequately addressed ways to improve pacemaker function. In this study we attempted to improve pacemaker function by altering connexin expression in rat mesenchymal stem cells (MSCs) to a phenotype similar to native sinus node pacemaker cells.</p><p><b>METHODS</b>To generate a biological pacemaker, MSCs were transduced with a cardiac pacemaker gene-hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), via transfection with a lentiviral vector. Funny current (I(f)) in HCN4(+) MSCs was recorded by voltage-clamp. Overexpression of connexin 45 (gene Gja7) in MSCs was achieved by transfection with the plasmid pDsRED2-N1-Gja7-RFP. Double-immunolabelling with anti-connexin 43 and anti-connexin 45 antibodies were used to identify the gap junction channels. The effects of the genetically modified MSCs on cardiomyocyte excitability were determined in MSCs cocultured with neonatal rat ventricular myocytes. Spontaneous action potentials of neonatal rat ventricular myocytes were recorded by current-clamp.</p><p><b>RESULTS</b>High level time- and voltage-dependent inward hyperpolarization current that was sensitive to 4 mmol/L Cs(+) was detected in HCN4(+) MSCs, confirming that HCN4 acted as I(f) channels in MSCs. Connexin 43 and connexin 45 were simultaneously detected in CX45(+) MSCs. Beating frequency was (82 +/- 8) beats per minute (n = 5) in myocytes cocultured with non-transfected control MSCs, versus (129 +/- 11) beats per minute (n = 5) in myocytes cocultured with HCN4(+) MSCs. Myocytes cocultured with MSCs cotransfected with HCN4 and connexin 45 had the highest beating frequency at (147 +/- 9) beats per minute (n = 5).</p><p><b>CONCLUSION</b>These findings demonstrate that overexpression of connexin 45 and subsequent formation of heteromeric connexin 45/connexin 43 gap junction channels in HCN4 expressing MSCs can improve their function as cardiac biological pacemakers in vitro.</p>

Epidemiology and surveillance programs on hemorrhagic fever with renal syndrome in Mainland China, 2005-2008 / 中华流行病学杂志

Objective To analyze the epidemiologic and surveillance data of hemorrhagic fever with renal syndrome (HERS) in China, from 2005 to 2008, to describe the epidemiology and trend of HERS. Methods Descriptive epidemiology were studied to analyze the surveillance data from 2005 to 2008, collected from both the internet-based national notifiable disease reporting system and 40 HFRS sentinel sites developed since 2005 in 40 counties around China. Results A total of 56 077 HERS cases and 692 deaths reported in China with case fatality rate as 1.23%. Morbidity and mortality had been annually decreasing since 2004. The top 7 provinces with HFRS cases were Heilongjiang, Lianning, Jilin, Shandong, Shaanxi, Hebei and Zhejiang, which had a total of 44 081 cases reported, accounting for 78.61% of the total number of cases, in the nation. More cases were reported in spring and autumn-winter season, with the peak in November. Cases reported in males were 3.13 times of the females and most cases seen in young and middle-aged farmers. The density and the virus carrying rate of animal hosts and the distribution of dominant species were relatively stable and similar to the previous findings. Apodemus agrarius and Rattus norvegicus were still the most common and predominant animal hosts. No genetic mutation of Hantavirus was detected in the surveillance program. Conclusion The continuous descending trend of the HERS epidemics could be related to the successful strategies on comprehensive prevention and control measures, as controlling the number of rodents and vectors, carrying out HERS vaccination campaign and health education by the local health care takers in the recent years. Implementation of the new national Expanded Program of Immunization on HFRS vaccine in high-risk areas may further reduce the epidemics. However,both the density and the virus carried rate among the host animals remain high in some areas, together with the emergence of new epidemic areas, all call for more attention to be paid on the disease.

Effects of volatie oil of Schizonepeta tenuifolia Briq herb and Saposhnikovia divaricata Schischke root on proinflammatory cytokine expression and regulation / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effects of volatie oil of Schizonepeta tenuifolia Briq herb and Saposhnikovia divaricata Schischke root (OSS) on proinflammatory cytokine expression and regulation in rats.</p><p><b>METHOD</b>OA and LPS were injected intravenously to rats to develop acute lung injury (ALI). The rats were treated with OSS (45.19 microL kg(-1)). The pathological sections of lung tissue were prepared and observed in acute lung injury rats. The expression of nuclear factor-kappa B p65 (NF-kappaB p65), intercellar adhesion molecule CD54, and NF-kappaB p65 mRNA were determined in lung cells.</p><p><b>RESULT</b>volatie oil of Schizonepeta tenuifolia Briq herb and Saposhnikovia divaricata Schischke root significantly inhibited the expression of CD54, the activation of NF-kappaB p65, and the transcription of NF-kappaB p65 mRNA.</p><p><b>CONCLUSION</b>OSS can reduce the expression of CD54 and NF-kappaB p65 protein synthesis, which may be its anti-inflammatory molecular mechanisms.</p>