AIM: To construct lentiviral vector carrying the angiopoietin-1(Ang-1) gene,and make it express Ang-1 in the rat mesenchymal stem cells(rMSCs).METHODS: The cDNA encoding the CDS of Ang-1 gene was obtained from the placenta of the adult Fisher 344 rats with RT-PCR.After digestion with restrication endonuclease,the Ang-1 gene was recombined to construct the transfer plasmid PNL-Ang1-IRES2-EGFP.The three-plasmid system of lentiviral vector was consisted of PNL-Ang1-IRES2-EGFP,the packaging plasmid HELPER,and the envelope plasmid VSVG,which were co-transfected to 293T cells mediated by lipofectamin2000 to produce lentiviral particles.The rMSCs were infected by obtained lentiviral particles.The insertion of Ang-1 gene was detected by PCR,the mRNA expression of Ang-1 in rMSCs was detected with RT-PCR,the protein expression of Ang-1 was observed with immunocytochemistry and Western blotting methods.RESULTS: The result of sequencing showed that the cloned Ang-1 gene was consistent with the sequence reported in GenBank.After digestion with restrication endonuclease,the 1 512 bp fragment of Ang-1 gene and the 10.5 kb vector fragment of PNL-IRES2-EGFP were observed with gel electrophoresis.The insertion of Ang-1 gene in viral genome was confirmed.The EGFP expression was observed with the fluorescent microscope.In infected rMSCs,the mRNA and protein expressions of Ang-1 were confirmed.CONCLUSION: Lentiviral vector carrying Ang-1 gene has been successfully constructed.The infected rMSCs are able to express the Ang-1 mRNA and Ang-1 abundantly.This will facilitate the following exploratory development of Ang-1 gene-modified rMSCs.

Objective To observe the effect of proanthocyanidin on the expression of nuclear factor kappa B (NF-кB) and interleukin-6 (IL-6) in rats with spinal cord injury (SCI). Methods 36 healthy adult Sprague-Dawley rats were randomly divided into proanthocyanidin treatment group (group A, n=12), methylprednisolone (MP) treatment group (group B, n=12) and model control group (group C, n=12). Allen's method was used to establish the model of acute spinal cord injury in T9. On the 1st, 3rd and 7th days after operation, 4 rats in each group were assessed with Basso-Beattie-Bresnahan (BBB) scale and Slanting Board Test (SBT), and the expression of NF-кB and IL-6 of the spinal cord were detected with immunohistochemistry. Results The scores of BBB and SBT were better in groups A and B than in the group C 3 and 7 days after SCI (P<0.05). Compared with group C, the expression of NF-кB in group A decreased all the time after SCI (P<0.01), and 3 and 7 days after SCI in group B (P<0.01); while the expression of IL-6 decreased all the time after SCI in groups A and B (P<0.05). Conclusion Proanthocyanidin may inhibit the expression of NF-кB and IL-6 in spinal cord after SCI in order to reduce inflammation and improve the hindlimb motor function in rats after SCI.

Objective To observe the effect of Proanthocyanidin on motor after spinal cord injury (SCI) in rats. Methods 36 healthy adult SD rats were divided into groups A, B and C (n=12), and SCI was induced with Allen's mode (250 g·mm) on T9. Proanthocyanidin 40 mg/kg was injected intraperitoneally for group A, methylprednisolone (MP) 30 mg/kg for group B and the same volume of saline for group C 30 min after SCI. 1 d, 3 d and 7 d after operation, all the rats were assessed with Basso-Beattie-Bresnahan (BBB) scale and slanting board test, and their serumal malondialdehyde (MDA) and superoxide dismutase (SOD) were detected. Results The scores of BBB and the slanting board test imporoved more in group A and group B than in group C (P<0.05). The SOD increased and MDA decreased in groups A and B significantly 1 d and 3 d after operation compared with those of group C (P<0.05), and only in group A 7 d after operation (P<0.05). Conclusion Proanthocyanidin may inhibit the lipid peroxidation and promote the recovery of motor after spinal cord injury in rats.

To construct the lentiviral RNA interference (RNAi) vector of rat CXCR4 gene, three target sequences were selected according to rat CXCR4 mRNA sequence, the complementary DNA contained both sense and antisense oligonucleotides were designed and synthesized. After phosphorylation and annealing, these double-strand DNA were cloned to Bgl II and Hind III sites of pSUPER. Then the product pRiCXCR4 was confirmed by electrophoresis and sequencing. Next, CXCR4 shRNA was cloned to a transfer vector of lentivirus, pNL-EGFP, and pNL-RiCXCR4-EGFP was produced. It was confirmed by digestion and sequencing that CXCR4 shRNA expression structure was correctly cloned to pSUPER and pNL-EGFP respectively. Three plasmids, pNL-RiCXCR4-EGFP, pHELPER and pVSVG were cotransfected into 293T to package lentivirus particles. The functional titer of obtained virus was determined by flow cytometry after transduction in 293T, the resulting functional titer of unconcentrated virus and concentrated virus were 6.4 x 10(4) TU/mL and 6.9 x 10(6) TU/mL respectively. After the rat mesenchymal stem cells (rMSCs) were transduced with the constructed lentiviral vectors, real-time RT-PCR, Western blotting and flow cytometry were used to evaluate the level of CXCR4 expression. Compared with control group, the CXCR4 mRNA expression were obviously suppressed in all three experimental groups (rMSCs-CXCR4a, rMSCs-CXCR4b, rMSCs-CXCR4c), especially the expression rate in rMSCs-CXCR4b group was reduced by 95.6%. The RNAi lentivirus vector of rat CXCR4 gene has been constructed successfully. This greatly facilitates the further studies such as evaluation the role of CXCR4 in rMSCs recruitment to damaged tissue.
Animals ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Lentivirus ; genetics ; metabolism ; Mesenchymal Stromal Cells ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; Rats ; Receptors, CXCR4 ; genetics ; metabolism ; Transduction, Genetic

Pretreatment with brain-derived neurotrophic factor (BDNF) reduces ischemic damage after focal cerebral ischemia, and bone marrow mesenchymal stem cells(MSCs) were reported to ameliorate functional deficits after stroke in rats. Here we investigate the synergistically therapeutic effects of BDNF gene-modified MSCs on cerebral infarction. We transfected MSCs with the BDNF gene using a lentivirus-based system and investigated whether the BDNF-modified MSCs contributed to improved functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model. Compared to untreated rats, rats that received both MSCs and BDNF-MSCs showed significantly more functional recovery. The difference in modified neurological severity score(mNSS) was statistically significant (P < 0.001). Recovery was better in BDNF-MSCs than in MSCs (P < 0.001). At the second week and second month after the systemic delivery of blank vector-modified MSCs and BDNF-modified MSCs, the treated rats exhibited more significant recovery than the control, including the accumulation and living of enhanced green fluorescence protein (EGFP)-positive cells in the infarct area and surrounding areas, neuron-like changes, expression of surface markers of neural cells, and a large amount of BDNF expression in the BDNF-MSCs-treated group. Our findings suggest that BDNF-gene-modified rMSCs can migrate to surrounding areas of the cerebral infarction lesion, differentiate into neural cells, and survive for extended periods. With the synergy of BDNF, they may promote the recovery of the neurological function following cerebral infarction and represent a new strategy for stem cell-based therapy.
Animals ; Brain-Derived Neurotrophic Factor ; biosynthesis ; genetics ; Genetic Vectors ; genetics ; Infarction, Middle Cerebral Artery ; genetics ; therapy ; Lentivirus ; genetics ; metabolism ; Male ; Mesenchymal Stem Cell Transplantation ; methods ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Rats ; Rats, Inbred F344 ; Recovery of Function ; Transfection

Generation of bio-engineered teeth by using stem cells will be a major approach for bioengineered implantation. Previous studies have demonstrated that dissociated tooth germ cells are capable of generating a tooth after reaggregation in vitro. However, the cellular and molecular mechanisms underlying this tooth regeneration are not clear. In this study, we dispersed E13.5 molar germ into single cells, immediately reaggregated them into cell pellet, then grafted the reaggregates under mouse kidney capsule for various times of culture. We investigated the morphogenesis and the expression of several developmental genes in dental epithelial cells in reaggregates of tooth germ cells. We found that dissociated tooth germ cells, after reaggregation, recapitulated normal tooth developmental process. In addition, dissociated dental epithelial cells retained the expression of Fgf8, Noggin, and Shh during reaggregation and tooth regeneration processes. Our results demonstrated that, despite of under dissociated status, dental epithelial cells maintained their odontogenic fate after re-aggregation with dental mesenchymal cells. These results provided important information for future in vitro generation of bio-engineered teeth from stem cells.
Animals ; Cell Culture Techniques ; methods ; Cell Differentiation ; Embryo, Mammalian ; Epithelial Cells ; cytology ; metabolism ; Female ; Gene Expression Regulation, Developmental ; genetics ; Male ; Mice ; Odontogenesis ; genetics ; Tooth Germ ; cytology ; physiology

Fast progresses in stem cell-based tooth tissue engineering have been achieved in recent years in several animal models including the mouse, rat, dog, and pig. Moreover, various postnatal mesenchymal stem cells of dental origin have been isolated and shown capable of differentiating into odontoblasts and generating dentin. Meanwhile, human keratinocyte stem/progenitor cells, gingival epithelial cells, and even iPSC-derived epithelium have been demonstrated to be able to differentiate into functional ameloblasts. Translational medicine studies in the nonhuman primate are irreplaceable steps towards clinical application of stem cell-based tissue engineering therapy. In the present study, we first examined the epithelial stem cell markers in the rhesus skin using immunostaining. Keratinocyte stem cells were then isolated from rhesus epidermis, cultured in vitro, and characterized by epithelial stem cell markers. Epithelial sheets of these cultured keratinocytes, which were recombined with E13.5 mouse dental mesenchyme that possesses odontogenic potential in the presence of exogenous FGF8, were induced to differentiate into enamel-secreting ameloblasts. Our results demonstrate that in the presence of appropriate odontogenic signals, rhesus keratinocytes can be induced to gain odontogenic competence and are capable of participating in odontogenesis, indicating that rhesus keratinocytes are an ideal epithelial cell source for further translational medicine study of tooth tissue engineering in nonhuman primates.
Ameloblasts* ; Animals ; Dentin ; Dogs ; Epidermis ; Epithelial Cells ; Epithelium ; Humans ; In Vitro Techniques ; Keratinocytes* ; Macaca mulatta ; Mental Competency ; Mesenchymal Stromal Cells ; Mesoderm* ; Mice* ; Models, Animal ; Odontoblasts ; Odontogenesis ; Primates ; Rats ; Skin ; Stem Cells ; Tissue Engineering ; Tooth ; Translational Medical Research

Background Exposure to air pollutants O₃ and PM_2.5 is closely related to population mortality. Most of the domestic research findings are for residents in coastal areas, and less for those in the central and western regions. Objective To investigate the acute effects of O₃ and PM_2.5 on the mortality of residents in a city of central China. Methods Data were collected on atmospheric pollutants, meteorological data, and population mortality in a city of central China from January 1, 2015 to June 30, 2021. Meteorological data included daily average temperature, air pressure, and relative humidity. Atmospheric pollution data included daily mean concentrations of PM_2.5, PM₁₀, SO₂, NO₂, and CO and maximum 8 h O₃. Generalized additive model with Poisson distribution was used for estimating the relationships between air pollutants (O₃ and PM_2.5) and population mortality, and further stratified by age, gender, and education. Results The daily maximum 8 h average concentration of O₃ in the city during the study period was 94.38 μɡ·m⁻³ and the daily average concentration of PM_2.5 was 55.56 μɡ·m⁻³. In the single-pollutant model, the correlations between O₃ concentration and total deaths as well as deaths due to respiratory, circulatory, hypertension, coronary heart disease, and stroke were strongest at lag02, lag2, lag02, lag0, lag02, and lag0, and for every 10 μɡ·m⁻³ increase in concentration of O₃, the associated ER (95%CI) values of daily mortality were increased by 0.09% (−0.08%–0.25%), 0.35% (0–0.71%), 0.43% (0.18%–0.68%), 0.45% (0.02%–0.91%), 0.59% (0.16%–1.02%), and 0.33% (0.01%–0.65%), respectively. The effect of O₃ on total mortality was not statistically significant (P>0.05). The correlations between PM_2.5 concentration and total deaths, as well as deaths due to respiratory, circulatory, hypertension, coronary heart disease, and stroke were strongest at lag1, lag5, lag01, lag05, lag04, and lag01, and for every 10 μɡ·m⁻³ increase in concentration of PM_2.5, the associated ER (95%CI) values of daily mortality increased by 0.02% (−0.09–0.13%), 0.25% (0.01%–0.50%), 0.35% (0.16%–0.54%), 1.18% (0.59%–1.77%), 0.17% (−0.13%–0.40%), and 0.65% (0.38%–0.92%), respectively, with no statistically significant effects of PM_2.5 on total mortality and mortality due to coronary heart disease (P>0.05). During warm season (from May to October), the ER (95%CI) values of total deaths per 10 μɡ·m⁻³ increase in O₃ in male, people aged 6~65 years, people aged >65 years, and people below high school education were 0.46% (0.16%–0.75%), 0.38% (0.08%–0.68%), 0.41% (0.14%–0.66%), and 0.38% (0.14%–0.61%), respectively, while the O₃ effect was not statistically significant (P>0.05) during cool season (from November to April). Conclusions Atmospheric pollutants (O₃ and PM_2.5) have acute effects on mortality in the city, with the elderly, people with less than a high school education, and those with circulatory disease being more sensitive to O₃ and PM_2.5 exposures.

Background In recent years, our country's atmospheric particulate matter pollution has improved significantly, while ozone (O₃) pollution has become increasingly serious. As a secondary pollutant, O₃ is closely related to human health. Objective To study the effect of short-term exposure to ozone in ambient air on population mortality in China. Methods A computer search with key words of "ozone or O₃", "death", and "time series" in Chinese or "ozone", "mortality", and "China" in English was performed in Web of Science, PubMed, China National Knowledge Infrastructure, Wanfang, and VIP databases to find literature on effects of short-term ozone exposure on population mortality covering a time period from January 1, 1990 to December 31, 2021. According to a set of inclusion and exclusion criteria developed for this study, literaturescreening, quality evaluation, andrelevant data extraction were carried out. Finally, R 4.1.2 software was used to perform meta-analysis to estimate target effect sizes. Results A total of 978 articles were retrieved. According to the inclusion and exclusion criteria, 18 articles were finally included, including 39 effect size estimates. The results showed that every 10 μɡ·m⁻³ increase in ambient ozone concentration was associated with an increase of 0.45% (95%CI: 0.39%-0.51%), 0.50% (95%CI: 0.33%-0.68%), and 0.60% (95%CI: 0.48%-0.72%) in total, respiratory, and cardiovascular disease mortalities , respectively. The results of subgroup analysis by age, sex, and season showed that when ozone concentration increased 10 μɡ·m⁻³, an increase of 0.34% (95%CI: 0.17%-0.51%) in mortality was observed in the ≥ 65-year-old population, higher than 0.09% (95%CI: −0.21%-0.39%) increase in the <65-year-old population; the mortality increase in females [0.44% (95%CI: 0.30%-0.58%)] was greater than that in males [0.35% (95%CI: 0.22%-0.48%)]; compared with the warm season [0.29% (95%CI: 0.16%-0.42%)], mortality increase was higher in the cold season [1.03% (95%CI: 0.71%-1.35%)]. Conclusion Ambient ozone is an important factor affecting population mortality. The elderly and women ≥ 65 years old in China are more sensitive to ozone, and the impact of ozone exposure on population mortality is greater in cold season.