Objective To evaluate the passage stability of HEK293 cells in suspension culture by using population doubling level(PDL)and verify the method,in order to provide experimental basis for the industrial production and culture of this cells. Methods The working seed lot of HEK293 cells were subcultured continuously for 60 d,one generation every 2 d,and the cell stability was evaluated when PDL increased to 10-60. The calculation of related research results showed that when HEK293 cells were cultured to 2 500 L,the PDL of each generation should be controlled within 2 ± 0. 2. The working seed lot of HEK293 cells were cultured in different stages of shaking flask(125,500,1 000 and 3 000 mL,four generations),cell expansion system(CES)(25,25 and 50 L,three generations)and bioreactor(100,500 and 500,three generations). The PDL of each generation was controlled within 2 ± 0. 2. Totally three batches of cells were cultured and analyzed for the indicators such as cell density,viability,agglomeration rate and diameter. After culture for 72 h in a 500 L bioreactor,the HEK293cells were inoculated with the working seed lot of adenovirus at a MOI of 5-10,cultured for 2 d,then the virus liquid was harvested and detected for the number of virus particles. Results HEK293 cells in the working seed lot in serial passage maintained high cell density and viability when the PDL reached 60. When PDL was controlled in the range of 2 ± 0. 2,the density of three batches of HEK293 cells in the shaking flask,CES and bioreactor was all greater than 2. 0 × 10~6cells/mL,the viability was all greater than 96%,and the cell diameter was about 17 μm. The agglomeration rates were all lower than 35%. The three batches of HEK293 cells cultured in the 500 L bioreactor were inoculated with virus for 2 d,and the number of virus particles reached 11. 68 × 1010,12. 55 × 10~(10)and 9. 38 × 10~(10)vp/mL,respectively. Conclusion It is feasible to evaluate the stability of passage of HEK293 cells by PDL,which can reflect the growth status of passage cells more scientifically.

BACKGROUND:Conventional dynamic hip screw or artificial joint replacement can be used to treat unstable intertrochanteric fracture in aged patients. It remains unclear whether we should select one-stage replacement or remedial joint replacement after failture, and there is no unified standard globaly. OBJECTIVE: To observe the outcomes and prognosis of one-stage artificial joint replacement for unstable intertrochanteric fracture in aged patients. METHODS:From April 2008 to October 2011, 21 patients with unstable intertrochanteric fracture in aged patients were repaired with one-stage artificial joint replacement at the Second Department of Orthopedics, Changji Prefecture People’s Hospital. Among 21 patients, 1 patient previously combined with avascular necrosis of the femoral head and traumatic arthritis received biological artificial total hip replacement. Three cases were subjected to standard bone cement bipolar artificial femoral head replacement. 17 cases underwent biological bipolar artificial femoral head prosthesis replacement. Al artificial joint, internal fixation material and accessory joint replacement surgical instruments were purchased outside China. Al patients were folowed up regularly. Hip joint function was assessed by Harris hip score. RESULTS AND CONCLUSION: Al operations were completed by the same group of physicians. Operation time was 30-60 minutes, averagely 42 minutes. Incision length was 8 to 15 cm, averagely 11 cm. Average intraoperative blood loss was 50-300 mL, averagely 150 mL. The number of transfusion cases was 13. 1.5 U blood was transfused averagely in each patient during hospital stay. One 76-year-old patient combined with hypertension, coronary heart disease and diabetes suffered from sudden death due to acute myocardial infarction at 9 days after replacement. B ultrasound revealed venous thrombosis of calf muscle of double lower extremities at 3 days after replacement. No complications such as prosthetic loosening, sinking, infections and thrombosis were detected. Except 1 case died, the other 20 cases received folow-up for 6-49 months. Harris hip score was 73±4 before discharge and 82±6 during last folow-up. These data confirm that effects of one-stage artificial joint replacement for unstable intertrochanteric fracture in aged patients are affirmative, but the number of case is stil less, and deserves further investigations. We suggest that in patients with conformed indication, one-stage artificial joint replacement can be the first choice.

BACKGROUND:Autologous peroneus brevis and alogeneic tendon are often used for reconstruction of lateral colateral ligament of the ankle joint, but these two kinds of materials have different histological and biomechanical properties. OBJECTIVE:To compare the clinical effects of autologous peroneus brevis and alogeneic tendon to reconstruct lateral colateral ligament of the ankle joint. METHODS: Sixty-six patients with chronic external ankle instability caused by old injury to lateral colateral ligament of the ankle joint were enroled, aged 15-63 years. The 34 of 66 patients underwent lateral ligament reconstruction using autologous peroneus brevis and the rest 32 patients received lateral ligament reconstruction using alogeneic tendon. After reconstruction, reaction time of the peroneous brevis, talar tilt angle and anterior talar translation, visual analog scale score and the American Orthopaedic Foot and Ankle Society (AOFAS) score were compared between the two groups. RESULTS AND CONCLUSION:At the last folow-up, the visual analog scale score, tilt angle and anterior talar translation were lowered in the two groups compared with the previous (P < 0.05), and the AOFAS scores were increased significantly in the two groups (P < 0.05); the reaction time of the peroneous brevis was increased inthe autologous peroneus brevis group (P < 0.05) and decreased in the alogeneic tendon group (P < 0.05); the above-mentioned indexes had no difference between the two groups. These findings indicate that autologous peroneus brevis and alogeneic tendon have similar effects on the lateral ligament reconstruction in terms of postoperative ankle function, stability and activity levels, but the alogeneic tendon shows advantages on less trauma and shorter operation time.

Objective To investigate the hemodynamic changes during the progression of portal hypertension(PHT) by establishing a model of liver cirrhosis in rats.Methods Totally 100 healthy male SD rats were assigned to 5 groups randomly(1 control group and 4 experimental groups with 20 rats in each group).Animal model of cirrhosis was established by subcutaneous injection of carbon tetrachloride(CCl4) and drinking alcohol.Rats in control group were given water and forage.The changes in the portal hemodynamics during the pathological process of liver tissues were observed after 2,4,7 and 10 weeks.Results During the formation of experimental cirrhosis,the hepatocytes of rats underwent 4 processes: degeneration,necrosis,fibrosis and pseudolobular proliferation.The hemodynamic changes were observed: the mean arterial pressure declined gradually after injection,but the portal venous pressure,the inferior vena cava pressure and the portal vascular resistance increased slowly.The portal venous flow reduced after ascending whereas the splanchnic vascular resistance increased after descending.Conclusion This method can establish a stable cirrhosis PHT model,which can be made in large quantities.During the progression of PHT,there are significant changes in portal vein hemodynamics and pathology,which can be used in related research on PHT.

Objective To compare prospectively the features and effects of combined operation(splenorenal shunt plus portal-azygous devascularization) and portal-azygous devascularization only(PCDV)on portal(hypertension)(PH).Methods We summarized 360 cases of PH admitted from 1984 to 2004.All patients were randomly divided into two groups,one was combined operative group(250 patients) and the other was PCDV group(110 patients).The therapeutic effects and changes of portal hemodynamics were studied with doppler flowmeter(DCFI),free portal pressure(FPP) and digital subtraction angiography(DSA) pre-and post-operatively,and were measured directly during the course of the procedure.Results(1)Postoperative bleeding:Of all the patients who underwent combined operation,no case of rebleeding occurred in the short period after operation,and the rebleeding rate was 8.0% in the long period of follow-up.In the patients who underwent PCDV,the rebleeding rate was 5.5% in the short period after operation,and 17.6% at long-term follow up(P0.05).(3)There was a significant decrease in the diameter of portal vein,and FPP postoperatively in the combined operation group compared to PCDV group.There was a significant decreases of PVF in the PCDV group.But the decrease of PVF in the two groups had no significant difference.Conclusions The combined procedure has merits of greater decrease of FPP,and alleviation of the condition of hyperdynamic blood flow in the portal vein.The clinical effect is also better than that of portal-azygous devascularization only.

Primary central nervous system lymphoma(PCNSL)is an aggressive extranodal non-Hodgkin's lymphoma involving cerebrospinal fluid,medulla spinalis,intraocular structures,cranial nerves,and pia.It is an infiltrating malignant tumor with no involvement outside the central nervous system.PCNSL is sensitive to radiotherapy and chemotherapy,and treatment includes high-dose methotrexate-based remission induction,surgery and consolidation therapy with whole-brain radiotherapy.In recent years,some new drugs,including Bruton tyrosine kinase inhibitor,programmed cell death protein-1 inhibitor,lenalidomide,rituximab,pemetrexed,chimeric antigen receptor T-cell immunotherapy and selective inhibitor of nuclear export,have entered clinical trials or use stage and shown good therapeutic effect.This article reviews the traditional treatment programs of PCNSL patients and summarizes the latest progress of treatment.

Cell-free protein expression system is a new method to express target protein in vitro and has been widely applied to the study of protein structure, protein function and other related fields. Preparation of cell extract is one of the key factors that affect the efficiency of the cell-free system. To improve the efficiency and economical feasibility of cell-free protein synthesis, we discussed the parameters during the preparation of the cell extract. These parameters include centrifugation speed, pre-incubation, and dialysis. We used the green fluorescent protein as the reporter protein, and obtained a simple procedure for the preparation of Escherichia coli cell extract. A simple centrifugation step (12 000 x g, 10 min) followed by a brief incubation was sufficient for the preparation of an active cell extract to support protein expression with higher productivity (209 microg/mL). Compared to the traditional E. coli S30 procedure, the processing time was reduced by 62%, and the productivity was increased by 2.6 times. The new procedure will make the advantage of cell-free technology more obvious, and promote its wider application.
Cell Fractionation ; methods ; Cell-Free System ; Escherichia coli ; cytology ; genetics ; metabolism ; Escherichia coli Proteins ; biosynthesis ; chemistry ; isolation & purification ; Green Fluorescent Proteins ; metabolism

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.

Objective:To investigate the effect of ureteral decellularized matrix (UDM) coating on the differentiation of adipose stem cells.Methods:From January 2018 to October 2019, UDM was prepared by perfusion method. H&E staining, DAPI staining, and DNA quantification were used to assessment the residues of cellular component in UDM and normal ureter; Masson′s trichrome staining and collagen quantification evaluated the collagen retention in UDM and normal ureter; the distribution and content of glycosaminoglycan in UDM and normal ureter were analyzed through Alcian Blue staining and glycosaminoglycan quantification. Canine adipose mesenchymal stem cells (cADMSCs) were isolated and cultured and identified by flow cytometry. The UDM was digested by pepsin enzyme to prepare the decellularized matrix coating as the experimental group. Type Ⅰ rat tail collagen coating was used as a control group. The cADMSCs were seeded on different coatings, and the differentiation of the cADMSCs was detected by immunofluorescence staining and Western Blot.Results:H&E and DAPI staining showed that the nuclear residue was not observed in the UDM. The DNA quantification demonstrated that the DNA content in UDM [(38.87±3.40) ng/mg] was significantly lower than that in the normal ureter group [(1 694.63±169.83) ng/mg, P<0.05]. Masson′s trichrome staining and collagen quantification confirmed that the collagen content in UDM [(265.89 ± 16.40) μg/mg] was no significantly different from the normal ureter group [(288.73 ± 16.32) μg/mg, P>0.05]. Alcian Blue staining showed the distribution of glycosaminoglycan in the UDM, and glycosaminoglycan quantification suggested that the content of glycosaminoglycan in the UDM [(1.57 ± 0.19) μg/mg] was lower than that in the normal ureter group [(3.43 ± 0.12) μg/mg] ( P<0.05). Immunofluorescence staining and Western Blot confirmed that the expression of Alpha-smooth muscle Actin (α-SMA) in the experimental group (2.51 ± 0.27, 3.68 ± 0.33, 4.91 ± 0.45) was higher than that in the control group (0.97±0.09, 1.02 ± 0.10, 1.00 ± 0.11) at 3 d, 7 d, 10 d ( P<0.05). The expression of α-SMA in the experimental group increased gradually with culture time ( P<0.05). While no changing of α-SMA expression in the control group was recordered. Conclusions:The prepared UDM removed the cellular components, and retained the collagen structure and bioactive components well; the ureter decellularized matrix coating could promote the differentiation of cADMSCs to smooth muscle cells.