Effect of Piezo1 on osteogenic differentiation of mouse bone marrow mesenchymal stem cells C3H10T1/2 based on CRISPR/Cas9

Objective·To investigate the effect of Piezo1 on osteogenic differentiation of mouse mesenchymal stem cells C3H10T1/2 cell line based on CRISPR/Cas9 system that can achieve stable gene knockout.Methods·According to the principle of CRISPR/Cas9 target design principle, two single guide RNAs (sgRNAs) were designed to construct lentivirus expressing Cas9 and lentivirus expressing sgRNA by using Lenti-Cas9-GFP and Lenti-U6-sgRNA-mCherry vectors. After the C3H101/2 cells were transfected with two types of lentiviruses, flow cytometry was used to screen mCherry- and GFP-positive cells. The monoclonal cells were selected, and amplified by PCR and agarose gel electrophoresis, and finally the monoclonal cell line with Piezo1 gene fragment knocked out was obtained. Sequencing, quantitative realtime PCR (qPCR) and immunofluorescence were performed to verify the the knockout efficiency of the constructed Piezol knockout C3H10T1/2 cells (CPK). CCK-8 assay was used to detect the effect of knocking out Piezo1 on cell proliferation; in vitro osteogenic induction differentiation was performed on successfully constructed Piezo1 gene knockout cells, and alkaline phosphatase (ALP) staining and alizarin red staining were used to investigate the effect of Piezo1 on osteogenic ability.Results·Positive clone was obtained in bacterial fluid of monoclonal cell lines with Piezol knocked out after PCR amplification and agarose gel electrophoresis. Sequencing analysis showed that a stop condon TGA was produced in exon 4 of Piezo1 gene in advance, so that the protein could not be translated correctly. qPCR verified that Piezo1 gene in CPK was inhibited at mRNA level; Immunofluorescence showed that the knockout efficiency of Piezo1 gene in CPK was high, which basically hindered the expression of Piezo1 in cells. CCK-8 assay showed that the cell proliferation ability decreased after knocking out Piezo1 (P<0.05); The results of ALP staining and alizarin red staining showed that the osteogenic ability of cells decreased after knocking out Piezo1(P<0.05). The mRNA expression levels of osteogenetic-related genes such as α 1 type Ⅰ collagen (Col1a1), Runt-related transcription factor 2 (Runx-2), osterix (Osx) and alkaline phosphatase (Alp) in CPK decreased significantly (all P<0.05).Conclusion·Piezo1 knockout C3H10T1/2 cells based on CRISPR/Cas9 system is constructed successfully and the osteogenic activity of stable Piezo1 knockout cell line is hindered significantly

The odontoblastic differentiation of dental mesenchymal stem cells: molecular regulation mechanism and related genetic syndromes

Dental mesenchymal stem cells (DMSCs) are multipotent progenitor cells that can differentiate into multiple lineages including odontoblasts, osteoblasts, chondrocytes, neural cells, myocytes, cardiomyocytes, adipocytes, endothelial cells, melanocytes, and hepatocytes. Odontoblastic differentiation of DMSCs is pivotal in dentinogenesis, a delicate and dynamic process regulated at the molecular level by signaling pathways, transcription factors, and posttranscriptional and epigenetic regulation. Mutations or dysregulation of related genes may contribute to genetic diseases with dentin defects caused by impaired odontoblastic differentiation, including tricho-dento-osseous (TDO) syndrome, X-linked hypophosphatemic rickets (XLH), Raine syndrome (RS), hypophosphatasia (HPP), Schimke immuno-osseous dysplasia (SIOD), and Elsahy-Waters syndrome (EWS). Herein, recent progress in the molecular regulation of the odontoblastic differentiation of DMSCs is summarized. In addition, genetic syndromes associated with disorders of odontoblastic differentiation of DMSCs are discussed. An improved understanding of the molecular regulation and related genetic syndromes may help clinicians better understand the etiology and pathogenesis of dentin lesions in systematic diseases and identify novel treatment targets

Weight of energy consumption parameters of rural residences in severe cold area

Appropriate management and optimization of energy consumption provides great support for the sustainable development of rural buildings. Although some research results are helpful for the energy-saving design and transformation of rural residential buildings, there is no systematic analysis of the impact of energy consumption parameters on energy-saving, which is not conducive to further optimization of energy-saving design. Based on more than 200 groups of field survey data, a typical initial rectangle model of rural residence in the severe cold area of northern China is established, and the orientation, aspect ratio, south window-wall ratio (WWR), north WWR, and enclosure structure are five main factors affecting the energy consumption in heating period. The method to evaluate the heating energy consumption of buildings was proposed based on EnergyPlus and analytic hierarchy process (AHP) from different functional parameters. The climate conditions of four cities of three secondary severe cold areas (1A, 1B, 1C) in northern China are selected for the building model, and the change rates and weights of energy consumption related parameters are analyzed. The results from the energy consumption simulation analysis show that the weight of enclosure structure is the largest, followed by the South WWR, the ratio of length to width is the third, and the orientation is the smallest in the four cities, which provides a good reference for the prediction and optimization of energy consumption in the rural residences in severe cold area

Does Gonadotropin Releasing Hormone Agonists plus add-back therapy bring an aurora to orthodontic treatment?

Introduction: Obviously, long therapy time of orthodontic treatment and a number of its adverse effects, such as pain, root resorption, enamel demineralization, periodontal disease, are the main reasons of complaints from patients. It is the first thing for an orthodontist to shorten the period of treatment and decrease the complications of orthodontic treatment as much as possible. The Hypothesis: We hypothesis Gonadotropin Releasing Hormone Agonists (GnRHa) and add-back therapy can create the "therapeutic window", namely, the appropriate estrogen level and assuage the adverse effects of estrogen deficiency which should be avoided as much as possible. Evaluation of the Hypothesis: It is generally acknowledged that estrogen has direct regulating role in bone metabolism by acting on osteoblasts and osteoclasts. Estrogen deficiency can increase the rate of orthodontic tooth movement and also bring about some adverse effects. The appropriate estrogen level, which we call the "therapeutic window" in orthodontic treatment, can speed up the orthodontic tooth movement and eliminate the adverse effects as far as possible. GnRHa can be the maker of estrogen deficiency; meanwhile, add-back therapy can remove the adverse effects by estrogen deficiency. So, we believe that GnRHa plus add-back therapy could be a new adjuvant method of orthodontic treatment and be good for orthodontists and patients

Osteoprotegerin and ligand of receptor activator of nuclear factor: kappaB expression in ovariectomized rats during tooth movement

Objective: To test the null hypothesis that increased tooth displacement in ovariectomized rats is not related to differential expressions of OPG and RANKL in the periodontium.\ud \ud Materials and Methods: Eighty-four 12-week female rats were used; half were ovariectomized and half were not. Three months later, the maxillary first molar was moved mesially. Groups of rats were sacrificed at days 0, 1, 3, 5, 7, 10, and 14 after activation. Tooth movement was measured at each time point. OPG and RANKL expressions were examined through immunohistochemistry.\ud \ud Results: Ovariectomized and nonovariectomized rats showed three-phase tooth movement. In both groups, OPG expression increased at the tension area and RANKL increased at the pressure area. The OPG/RANKL ratio coincided with tooth movement, especially in the linear phase from 7 to 14 days.\ud \ud Conclusions: The null hypothesis is rejected. The increased rate of tooth movement in ovariectomized rats was related to differential expressions of OPG and RANKL