Objective To compare the effects of three kinds of fixative solutions on paraffin section of mouse lens tissue and optimize the fixing?method of paraffin section in mouse lens tissue. Methods Three kinds of conventional fixatives were selected for the test ,including the conven?tional Davison’s solution,modified Davison’s solution and 10%neutral buffered formalin. Mice eyeball tissues were fixed with three different fixa?tives,embedded,sliced and then stained with HE method. The paraffin slices were observed under the light microscope. Results The structures of lens and retina fixed in conventional Davison ’s fixative solutions were clear and intact ,and the cells were arranged regularly and compactly. There was no eyeball distortion,contraction and retinal detachment in the eyeballs fixed in modified and conventional Davison’s fixative solution. However,the ones fixed in 10%neutral buffered formalin showed eyeball distortion and contraction,space and spherules. Conclusion The mice lens slides made from tissues fixed by conventional Davison ’s fixative solution are better than fixed by modified Davison ’s fixative solution and the 10%neutral buffered formalin fixed ones.

Objective To explore expression and clinical value of VEGF-C and p63 in early esophageal carcinoma and intraepithelial neoplasia. Methods 146 cases were randomized into normal esophageal mucosa, low level intraepithelial tumor, high level intraepithelial tumor and early esophageal carcinoma. The expression of VEGF-C and p63 were detected by using the immunohistochemistry dyeing.Results The expression of VEGF-C immunohistochemistry dyeing had statistical differences among different levels(X~2= 47.455, P <0.001). Normal esophageal mucosa v.s. high level intraepithelial tumor (X~2=36.721, P <0.001), Normal esophageal mucosa v.s. early esophageal carcinoma (X~2=26.483, P <0.001), low level intraepithelial tumor v.s. high level intraepithelial tumor(X~2= 10.025, P<0.0083), low level intraepithelial tumor v.s. early esophageal carcinoma(X~2=16.734, P<0.001). There was a significant correlation between pathological classification and the expression amount of VEGF-C (r = 0.462, P <0.001). The expression of p63 had statistical differences among different levels(X~2=28.962, P <0.05). There was a significant difference on normal esophageal mucosa comparing with low level, high level intraepithelial tumor or early esophageal carcinoma (X~2=12.735, P =0.005, X~2=20.421, P<0.001, X~2=20.854, P<0.001). There was a significant correlation between pathological classification and the expression of p63 (r= 0.272, P<0.05). Conclusion There is a significant correlation in the express of either VEGF-C or p63 comparing with either intraepithelial tumor or early esophageal carcinoma. It may be an early warning indicator.

Objective To explore the effects of using proton pump inhibitors (PPIs) on the outcomes of hip fracture.Methods Searches were conducted through Medline,Embase,Cochrane Library and Chinese Biomedical Literature Database to identify the studies of the association between PPIs exposure and hip fracture.Quality of studies was assessed using the Newcastle-Ottawa Quality Assessment Scale.Pooled odds ratios (ORs) and 95％ confidence interval (CIs) were calculated for the risk of hip fracture associated with current exposure of PPIs.And several subgroups were analyzed by dosing duration,dose,osteoporosis and corticosteroid usage to explore potential study heterogeneities.All statistical analyses were performed with STATA software.Results Among 11 publications included for final analysis,there were a total of 1 107 577 subjects with an average age of over 60 years.A positive relationship existed between PPIs exposure and hip fracture with an OR of 1.46 (95％ CI:1.26-1.70,P =0.000) as compared with nonPPI-users,especially those on concurrent corticosteroid and PPIs.A significantly increased risk of hip fracture was found in the group of a short-term duration for under 1 year (OR =1.18,95％ CI:1.01-1.38,P =0.041),medium-term for 1-3 years (OR =1.23,95％ CI:1.01-1.49,P =0.038) and longer duration for over 6 years (OR =1.38,95％ CI:1.27-1.50,P =0.000).Furthermore,concurrent use of PPIs was not associated with an increased risk of hip fracture in a definite dose-response manner.As compared with non-PPI-users,no significantly increased risk of hip fracture was found in PPI-users with osteoporosis (P ＞ 0.05).Publication bias was not present.Conclusions Use of PPIs may be somewhat associated with an increased risk of hip fracture.Considering potential adverse effects,clinicians should prescribe cautiously PPIs for high-risk patients,especially elders.

The kidney stores essence,essence generates marrow is one of the important contents in zaag-xiang theory of traditional Chinese medicine (TCM).The kidney-marrow-brain,kidney-marrow-bone and other biological axis put forward by modern scholars took kidney-marrow system as their cores.Derived from this basis as well as under the guidance of TCM holism concept,the construction of TCM kidney-marrow system has been continuously improved;and the kidney-marrow correlation theory has been enriched.To better grasp and apply the kidney-marrow correlation theory can solve clinical problems of major diseases.However,doctors in past dynasties did not clearly define kidney-marrow system. It has not formed a complete theoretical system.Marrow in the kidney-marrow system contained brain,spinal cord and bone marrow.Marrow can generate blood,which nourishes bone together with the bone marrow.The bone marrow connects with the brain and spinal cord,which are all related to blood.Hence,the function of marrow generates blood occupied an important position in the kidney-marrow system. In this paper,the structure and function of the kidney-marrow system were preliminarily explained and constructed;and the kidney-marrow system related theories were further enriched.

There was preliminary understanding of the kidney-marrow correlation from the pre-Qin to Han dynasty.Until Wei-Jin and Sui-Tang dynasty,the close relationship between kidney-essence-marrow-bone-blood-brain was gradually realized in terms of the structure and physiological function.There was a new anatomical understanding of marrow in the Song,Jin and Yuan dynasty.The concept of spinal cord was put forward for the first time.There was also the etiology and pathogenesis on kidney marrow related diseases.In the Ming and Qing dynasty,there was in-depth understanding on the kidney-marrow correlation.However,the concept of kidney-marrow system was not clearly proposed.Modern scholars summarized traditional Chinese medicine (TCM) theories on kidney stores essence,essence generates marrow,marrow enriches brain,nourishes bone and transfers into blood. Then,the kidney-marrow systemhad begun to take form.It laid a theoretical foundation for the construction of kidney-marrow system..

Through reviewing and summarizing classical literatures of traditional Chinese medicine (TCM) and modern medical research results,TCM kidney-marrow system was presented and initially explained.The kidney marrow system is inseparable in structure,interdependent in physiology and mutually influential in pathology.The kidney-marrow system can be applied to the treatment of major diseases in TCM,which included metabolic bone diseases,brain and neurological disorders,haematological diseases and etc.,with significant clinical effects.The in-depth study on TCM kidney marrow system will improve the clinical curative effect of major TCM clinical diseases and achieve the precision treatment in traditional medicine.It displays unique advantages of TCM holism concept and achieves new breakthrough in TCM clinical diseases.

Objective: To construct ¹³¹I-the fifth generation polyamidoamine (PAMAM(G5.0)) with targeting peptide Ser-Arg-Glu-Ser-Pro-His-Pro (SRESPHP; SR) or Gly-Pro-Leu-Pro-Leu-Arg (GPLPLR; GP) and double targeting peptide SR/GP, and evaluate the targeting ability in medullary thyroid carcinoma (MTC) model. Methods: PAMAM(G5.0), PAMAM(G5.0)-SR, PAMAM(G5.0)-GP and PAMAM(G5.0)-SR/GP were radiolabeled with ¹³¹I by chloramine T method. The radiolabeled yield and radiochemical purity were determined by thin layer chromatography. MTC xenografts were developed and the percentage radio-activity of injection dose per gram of tissue (%ID/g) in tumor and organs was measured at 24 h post-injection. Region of interest (ROI) was drawn and the tumor/non-tumor (T/NT) ratios at 4, 8 and 24 h post-injection were calculated and compared among different groups. One-way analysis of variance, repetitive measurement analysis of variance and Dunnett-t test were used to compare the data of different groups. The relationship between %ID/g and T/NT was analyzed with Pearson correlation. Results: The radiolabeled yield was more than 75% and radiochemistry purity was more than 90%. The difference of %ID/g at 24 h post-injection was significant (F=14.400, P<0.001) in tumors of all groups. The radioactive uptake in tumor of ¹³¹I-PAMAM(G5.0)-SR group was the highest at 24 h post-injection((1.80±0.18) %ID/g). There were significant differences of T/NT ratios among different groups(F=4.776, P<0.05)and between different time points(F=8.630, P<0.05). Compared with negative control group (Na¹³¹I), the T/NT ratios significantly increased in ¹³¹I-PAMAM(G5.0)-SR group at 4, 8 and 24 h post-injection (t=4.169, 7.123 and 4.032, all P<0.05) and in ¹³¹I-PAMAM(G5.0)-GP group at 4 h post-injection (t=5.893, P<0.05). The T/NT ratio in ¹³¹I-PAMAM(G5.0)-SR group was higher than that in ¹³¹I-PAMAM(G5.0)-GP group at 24 h post-injection (t=2.871, P<0.05). Conclusions PAMAM(G5.0)-SR, PAMAM(G5.0)-GP and PAMAM(G5.0)-SR/GP can target the MTC models. ¹³¹I-PAMAM(G5.0)-SR has the best biological properties and may provide a new precision method for MTC diagnosis, treatment and prognosis evaluation.

Objective:To investigate the effect of tissue engineered bladder patch constructed by double-layer silk scaffold and adipose-derived stem cells (ADSCs) in the repair and reconstruction of bladder.Methods:This study was conducted from May 2020 to March 2021. The silk fibroin (SF) aqueous solution was obtained from silkworm cocoons, and a double-layer silk scaffold composed of silk fibroin film and silk fibroin sponge was further prepared. The rat ADSCs were isolated, cultured, and the ADSCs surface markers (CD29, CD90, CD45, CD106) were identified by flow cytometry. The ADSCs were planted on a double-layer silk scaffold to construct a tissue-engineered bladder patch. Thirty-six male SD rats were randomly divided into three groups: tissue engineered bladder patch group (SF-ADSCs group, n=15), double-layer silk scaffold group (SF group, n=15), control group ( n=6). The tissue engineered bladder patch (SF-ADSCs group) and double-layer silk scaffold (SF group) were wrapped on the omentum to promote vascularization. The vascularization was evaluated by HE and immunofluorescence staining. The wrapped tissue engineered bladder patch and double-layer silk scaffold were used to repair the defective bladder. In the control group (six rats), the incision was closed immediately after the bladder tissue fully exposed. At 4 weeks and 12 weeks after operation, the general morphology of bladder tissue and cystography were performed to evaluate the recovery of bladder morphology. After the graft was harvested, HE and Masson's trichrome staining and immunofluorescence staining were used to observe the regeneration of bladder wall tissue. Urodynamics was used to assess the recovery of bladder function at 12 weeks after operation. Results:The flow cytometry results confirmed that the isolated cells positively expressed CD29 and CD90, and there was no significant expression of CD45 and CD106. Gross observation and scanning electron microscope confirmed that the preparation of double-layer silk scaffold not only had a pore structure that was conducive to cell planting, but also had good toughness and was facilitated to surgical suture. The number (43.50±2.66) and area (0.73±0.03)% of vascular-like structures in the SF-ADSCs group after the omentum encapsulation was significantly higher than that in the SF group [(24.50±3.51), (0.55±0.05)%], and the difference was statistically significant ( P<0.05). At 4 weeks after bladder repair, the histological staining of the grafts in the SF-ADSCs and SF groups showed a large number of degraded fragments of double-layer silk scaffold. At 12 weeks, the morphology of the graft in the SF-ADSCs group showed uniform bladder morphology, which was similar to that of normal bladder tissue. Immunofluorescence staining showed that the continuous urothelial layer, abundant smooth muscle tissue, vascular structure and regenerated neurons could be observed in the SF-ADSCs group. Urodynamic test showed that the bladder maximum volume (0.74±0.03)ml and compliance (16.68±0.44)μl/cm H 2O in the SF-ADSCs group, which were better than that in the SF group [(0.47±0.05)ml, (14.89±0.37)μl/cm H 2O], but lower than that in the control group [(1.12±0.08)ml, (19.34±0.45)μl/cm H 2O], and the difference was statistically significant ( P<0.05). Conclusions:The tissue engineered bladder patch constructed with double-layer silk scaffolds and ADSCs could promote the morphological repair of bladder tissue, the regeneration of bladder wall structure and the recovery of bladder physiological function.

Objective:To prepare the whole bladder acellular matrix (BAM) using the self-designed perfusion decellularization system, and evaluate the feasibility of constructing the tissue engineering bladder with the adipose-derived stem cells (ADSCs).Methods:This study was conducted from October 2020 to April 2021. The self-designed perfusion decellularization system was used, and four different decellularization protocols (group A, group B, group C and group D) were formulated, according to the flow direction of the perfusate and the action time of different decellularization solutions. Among them, the urethral orifice of the bladder tissue was used as the outflow tract of the perfusion fluid in groups A and B. The top of the bladder was cut off and used as the outflow tract of the perfusion fluid in groups C and D. In groups A and C, 1% Triton X-100 was treated for 6 h, and 1% sodium dodecyl sulfate (SDS) was treated for 2 h. In groups B and D, 1% Triton X-100 was treated for 7 h, and 1% sodium dodecyl sulfate (SDS) was treated for 1 h. In addition, the tissue in the normal bladder group was directly obtained from the natural bladder tissue, which did not require perfusion, cryopreservation and thawing. The fast and efficient decellularization protocol was screened out through HE, DAPI, Masson trichrome and Alcian Blue staining and quantitative analyses to prepare the whole bladder scaffold. The prepared BAM was used as the scaffold material, and the ADSCs were used as the seeding cells to construct the tissue engineering bladder. HE and DAPI staining were used to observe the distribution of ADSCs on the BAM.Results:HE and DAPI staining showed that there was no obvious nuclear residue in the group C. Masson trichrome and Alcian Blue staining showed that the collagen structure and glycosaminoglycan were well preserved in the group C. There was no significant difference in bladder wall thickness between the group C and the normal bladder group [(975.44±158.62)μm vs.(1 064.49±168.52)μm, P > 0.05]. The DNA content in the group C [(43.59 ±4.59) ng/mg] was lower than that in the normal bladder group, group A, group B and group D [(532.50±26.69), (135.17±6.99), (182.49±13.69) and(84.00±4.38)ng/mg], and the difference was statistically significant ( P<0.05). The collagen content [(10.98 ± 0.29)μg/mg] and glycosaminoglycan content [(2.30±0.18)μg/mg] in group C were not significantly different with those in the normal bladder group [(11.69±0.49) and (2.36±0.09)μg/mg, P>0.05]. Scanning electron microscopy showed that a large number of pore structures could be observed on the surface of the prepared BAM in groups A-D and were facilitated to cell adhesion. The isolated and cultured ADSCs were identified by flow cytometry to confirm the positive expression of CD90 and CD29, and the negative expression of CD45 and CD106. Live/dead staining and CCK-8 detection confirmed that the prepared BAM in the group C had no cytotoxicity. HE and DAPI staining showed that a large number of ADSCs were distributed on the surface and inside of the tissue engineering bladder. Conclusions:The whole bladder shape BAM prepared by the self-designed perfusion decellularization system could be used as the scaffold material for bladder tissue engineering, and the constructed tissue engineering bladder could be used for bladder repair and reconstruction.

To study the effect of the Tangshenping containing serum on the proliferation of the high glucose-induced epithelial cells of renal tubules.To prepare durg-contained serum from rats to enter into in vitor reaction system,the cellscultured via 10％ FBS-RPMI 1640 were randomly divided into 7groups:the normal group,themodel group,the Y27632 group,theirbesartangroup,the small dose Tangshenping group,the medium dose Tangshenping group and the high dose Tangshenping group and The cells were cultured in 3000 cells/well and grown in 96-well plates.Each group had 8 wells,then detect the effects of all serum sections on the proliferation of high glucose-induced epithelial cells of kidney tubules by the MTT colorimetric method after cultured for 12 h,24 h,48 h,and 60 h.Based on the results above,cell protein were extracted from each group at 24 h,and the expression of RhoA,ROCK1,α-SMA and E-cadherin in each group were detected by Western blotting.After high glucose stimulation,the shape of cell was shuttle-like or irregular triangle,the way it grew was radial;after the intervention of the corresponding serum,the shape of the cell was fiat and irregular polygonal.Started with 12h,compared with the normal group,OD value of other groups increased;at the 24h、48hand 60h,compared with the normal group,OD value of high glucose groupincreased significantly (P＜0.01);compared with the high glucose group,OD value of treatment groups decreased (P＜0.05);and 48 h,compared with the Y27632group,irbesartan groupand Tangshenping high dose group,OD value of Tangshenping low and medium dose groups decreased (P＜0.05);60 h,compared with Y27632 group,OD value of Tangshenping medium dose groups decreased;compared with irbesartan group,OD value of o Tangshenpinggroupsdecreased (P＜0.05);compared with Tangshenping high dose group,OD value of Tangshenpinglow groupsdecreased (P＜0.05) Western blotting analysis showed that compared with normal group,the expression of E-Cadherin protein in high glucose group reduced,and the expression of RhoA,ROCK1 and α-SMA protein increased;compared with high glucose group,the expression of E-Cadherin protein in each treating group increased,and the Tangshenping large dose group wassignificantly different (P＜0.01);the expression of RhoA,ROCK1 and or-SMA protein reduced,Tangshenping,the large dose group was significantlydifferent (P＜0.01);Compared with the Y27632 group,the expression of E-cadherin,ROCK1 and α-SMA protein in Tangshenping large dose group had no significant difference,while the expression of RhoAproteinreduced (P ＜0.01).Compared with theirbesartan group,the expression of E-cadherin,RhoA,ROCK1 and α-SMA protein in Tangshenping large dose group had no significant difference (P＞0.05).The Tangshenping containing serum is abletoinhibit the proliferation of high glucose-induced epithelial cells of kidney tubules,and can reverse renal tubular-epithelial cell transdifferentiation via regulating RhoA/ROCK signaling pathway,and restrain renal interstitial fibrosis,thereby delaying the pathogenesis of diabetic kidney disease.