0.05), but in the HP + group, the D cells markedly reduced as compared with HP - group and showed a significantly difference(P

Objective:Human adipose-derived stem cells (hASCs)are a highly attractive source in bone tissue engineering.To generate a luciferase reporter system that could be used to quantitatively and rapidly examine osteogenic differentiation potential of human adipose-derived stem cells (hASCs ) in vitro,and eventually make it possible to monitor the osteogenic differentiation of transplanted cells in vi-vo.Methods:The genomic DNA harboring promotor regions of osteocalcin and DNA sequences encoding luciferase genes were amplified by PCR and cloned into the pLVX-pTRE-puro vector to generate the OCpro-Luc-Puro construct.Then,the OCpro-Luc-Puro construct together with three assistant vectors:pM-DLg/pRRE,pRSV-REV,and pVSVG,were transiently transfected into HEK293T cells followed by viral supernatants collection,filtration and concentration.Next,the hASCs stably expressing luciferase repor-ter gene driven by osteocalcin promotor were created with the lentivirus carrying OCpro-Luc-Puro cassette under puromycin selection.The OCpro-Luc-hASCs were then cultured in the absence or presence of osteo-genic differentiation medium.On the 7th and 1 4th days,after osteogenic induction,cellular extracts were collected and analyzed by luciferase reporter assay.Meanwhile,alizarin red staining and quantification as well as quantitative reverse transcription PCR (qRT-PCR)analysis of osteogenic associated genes osteo-calcin (OC),runt-related transcription factor 2 (Runx2)and alkaline phosphatase (ALP)were used to assess the osteogenic differentiation ability of OCpro-Luc-hASCs.Results:OCpro-Luc-Puro plasmid and OCpro-Luc-hASCs were successfully generated.On the 7th and 1 4th days after osteogenic induction,the luciferase activity of the cellular extracts from OCpro-Luc-hASCs was dramatically increased.Consistently, the extracellular matrix mineralization,as shown by Alizarin red S (ARS)staining and quantification was also markedly intensified and a marked increase of the mRNA expression levels of OC,Runx2 and ALP, although to variable extent,was observed upon osteogenic differentiation.These results indicated that the observations from traditional experiments examining hASCs osteogenic differentiation were largely in agreement with that of our luciferase reporter assay in OCpro-Luc-hASCs.Conclusion:We established a luciferase reporter system that could be used to rapidly,quantitatively and specifically determine osteo-genic differentiation ability of hASCs.Comparing with the traditional time-consuming methods,the system we generated here was highly effective.This system not only can be used to examine ostogenic differentia-tion of hASCs in a high throughput manner,but also provides a way to monitor ostogenic differentiation of cells in vivo.

This study aimed to explore the mechanism of volatile oil from purslane in treating itch induced by eczema through establishing the itch model,stimulating the keratinocyte with capsaicine (CAP).SD rats were divided into the control group,the model group,the high dose group of volatile oil from purslane,and low dose group of volatile oil from purslane.After finishing the experiment,the morphology of keratinocytes was observed by immunofluorescence technique,while Ca2+ concentration was detected by flow cytometry,and the contents of leukotriene A4 methyl ester (LTA4),interleukin-31 (IL-31) and hydroxy trptamine H1 (HTH1) were quantified by ELISA assay.The expression of TRPV1 mRNA in keratinocytes was tested by RT-PCR,while the protein level of TRPV1 was quantified by western blot.Compared with the model group,it was found that the cell count of positive keratinocytes,the Ca2+ concentration,the levels of LTA4,IL-31 and HTH1,and the mRNA and protein expressions of TRPV1 in the high dose and low dose groups were significantly decreased (P ＜ 0.05).In conclusion,it was demonstrated that the volatile oil from purslane may relieve itch through inhibiting the activation of TRPV1 and reducing secondary inflammatory reaction.

Objective:To investigate the influence and mechanism of Portulaca extracts on Tumor Necrosis Factor -α(TNF-α), Interleukin-4(IL-4) in skins of rats with atopic eczema (AE).Methods:40 SD rats were randomly divided into normal control group , model group,Cortex Phellodendri group and Porulaca group.2,4-dinitrochlorobenzene was used on the right back of rat for making atopic eczema.21 days later , all animals were sacrificed , compared the weight of the same size of the eczema and healthy skin.The levels of TNF-αand IL-4 in rats skins were detected by immunohistochemical method.Results:The right back of rats in model group appeared obvious erythema ,erosion and leakage ,which meant the modeling succeeded.Compared with model group ,the swelling degree of Cortex Phellodendri group and Purslane group were significantly decreased ( P<0.01 ) ,compared with Cortex Phellodendri group ,the swelling degree of Purslane group were less than them ( P<0.05 ).The levels of TNF-α, in rats skins of normal control group , model group,Cortex Phellodendri group and Portulaca group were (6 652.66±1 190.94 ),(19 927.10±5 494.21 ), (7 515.13±877.66 ), (6 809.93±1 385.54).The levels of IL-4 were (5 378.44±1 685.01),(26 334.89±3 993.48),(7 814.84±1 751.38),(8 246.57± 975.08 ).Compared with model group ,the levels of TNF-α,IL-4 in rats skins of Cortex Phellodendri group and Purslane group were sig-nificantly decreased(P<0.01).Compared with positive drug group,the levels of TNF-α,IL-4 in rats skins of Purslane group were a little decreased(P<0.05).Conclusion:Purslane extracts has effect to treat atopic eczema ,its possible mechanism is by erducing the proinflammatory factor ,and moderating the anti-inflammatory factor and the ratio between them.

Objective:To explore the application effect of dyadic intervention scheme based on dyadic disease management theory and Information, Knowledge, Attitude, and Practice model in the discharge preparation of elderly stroke patients and family caregivers.Methods:The 92 pairs of elderly stroke patients and their caregivers hospitalized in the Department of Neurology in People′s Hospital of Zhengzhou University were conveniently selected. The non synchronous control method quasi experimental research was adopted. Totally 46 pairs of subjects who met the criteria for admission and discharge from May to July 2022 were set as the control group, and routine nursing was carried out; from August to October 2022, 46 pairs of subjects who met the criteria for admission and emission were set as the observation group to implement the dyadic intervention program. The scores of discharge readiness, self-efficacy and unplanned readmission rate of patients between the two groups were compared, and the scores of caregiver readiness, self-efficacy and caregiver stress between the two groups were compared.Results:Finally, 85 pairs of subjects completed the study, with 42 pairs in the control group and 43 pairs in the observation group. On discharge day, the total scores of discharge readiness and caregiver readiness in the observation group were (95.19 ± 4.47), (23.02 ± 2.20) points, respectively, which were higher than those in the control group (85.71 ± 5.31), (19.57 ± 1.65) points, with statistically significant differences ( t=8.91,8.16, both P<0.01); the self-efficacy levels of patients in the observation group at discharge and one month after discharge, as well as those of caregivers at discharge and one month after discharge were (73.86 ± 4.87), (75.91 ± 4.51), (75.67 ± 4.99), (79.21 ± 4.90) points, respectively, higher than those in the control group (71.62 ± 5.19), (73.33 ± 4.91), (73.48 ± 4.24), (75.48 ± 4.24) points, with statistically significant differences ( t values were from 2.05 to 3.75, all P<0.05); the pressure levels of caregivers in the observation group at discharge and one month after discharge were (7.51 ± 2.48), (6.28 ± 1.99) points, respectively, lower than those in the control group (8.76 ± 2.55), (7.45 ± 2.36) points, with statistically significant differences ( t=-2.29, -2.48, both P<0.05); the unplanned readmission rate of patients in the observation group one month after discharge was 7.0% (3/43), lower than the control group′s 23.8% (10/42), with statistically significant difference ( χ2=4.65, P<0.05). Conclusions:The implementation of dyadic intervention on elderly stroke patients and caregivers can make their discharge preparation process more adequate, thus reducing the caregiver′s care pressure, reducing the unplanned readmission rate of patients, and improving their health outcomes.

Objective:To explore the effect of ubiquitin-specific protease 42(USP42)on osteogenic differentiation of human adipose-derived stem cells(hASCs)in vivo and in vitro.Methods:A combina-tion of experiments was carried out with genetic depletion of USP42 using a lentiviral strategy.Alkaline phosphatase(ALP)staining and quantification,alizarin red S(ARS)staining and quantification were used to determine the osteogenic differentiation ability of hASCs under osteogenic induction between the experimental group(knockdown group and overexpression group)and the control group.Quantitative re-verse transcription PCR(qRT-PCR)was used to detect the expression levels of osteogenesis related genes in the experimental group and control group,and Western blotting was used to detect the expression levels of osteogenesis related proteins in the experimental group and control group.Nude mice ectopic im-plantation experiment was used to evaluate the effect of USP42 on the osteogenic differentiation of hASCs in vivo.Results:The mRNA and protein expressions of USP42 in knockdown group were significantly lower than those in control group,and those in overexpression group were significantly higher than those in control group.After 7 days of osteogenic induction,the ALP activity in the knockdown group was sig-nificantly higher than that in the control group,and ALP activity in overexpression group was significantly lower than that in control group.After 14 days of osteogenic induction,ARS staining was significantly deeper in the knockdown group than in the control group,and significantly lighter in overexpression group than in the control group.The results of qRT-PCR showed that the mRNA expression levels of ALP,os-terix(OSX)and collagen type Ⅰ(COL Ⅰ)in the knockdown group were significantly higher than those in the control group after 14 days of osteogenic induction,and those in overexpression group were signifi-cantly lower than those in control group.The results of Western blotting showed that the expression levels of runt-related transcription factor 2(RUNX2),OSX and COL Ⅰ in the knockout group were significant-ly higher than those in the control group at 14 days after osteogenic induction,while the expression levels of RUNX2,OSX and COL Ⅰ in the overexpression group were significantly lower than those in the control group.Hematoxylin-eosin staining of subcutaneous grafts in nude mice showed that the percentage of osteoid area in the knockdown group was significantly higher than that in the control group.Conclusion:Knockdown of USP42 can significantly promote the osteogenic differentiation of hASCs in vitro and in vi-vo,and overexpression of USP42 significantly inhibits in vivo osteogenic differentiation of hASCs,and USP42 can provide a potential therapeutic target for bone tissue engineering.