[Comparison of open reduction hollow nail anchoring system with loop plate fixation under arthroscopy for the treatment of posterior cruciate ligament avulsion fractures].

OBJECTIVE: To compare clinical effect between open reduction and fixation with cannulated screw and threaded rivet via posteromedial approach versus arthroscopic Endobutton plate fixation in treating posterior cruciate ligament avulsion fractures. METHODS: Clinical data of 38 patients with posterior cruciate ligament avulsion fractures from July 2020 to December 2021 were analyzed retrospectively, and divided into open reduction and internal fixation group (posterior medial approach hollow anchor system fixation) and arthroscopic fixation group (Endobutton with loop plate fixation under arthroscopy). There were 20 patients in open reduction and internal fixation group, including 16 males and 4 females, aged from 26 to 74 years old with an average of (42.9±18.8) years old;13 patients on the left side and 7 patients on the right side;12 patients were classified to typeⅡand 8 patiens with type Ⅲ according to Meyers-McKeever fractures classification;14 patients were gradeⅡand 6 patients were grade Ⅲ in back drawer test. There were 18 patients in arthroscopic fixation group, including 11 males and 7 females;aged from 24 to 70 years old with an average of (53.5±13.4) years old;11 patients on the left side and 7 patients on the right side;10 patients were classified to typeⅡand 8 patiens with type Ⅲ according to Meyers-McKeever fractures classification;11 patients were gradeⅡand 7 patients were grade Ⅲ in back drawer test. Operation time, blood loss, and quality of immediate reduction were compared between two groups. Knee range of motion, knee back drawer test, and International Knee Documentation Committee(IKDC) grading, KT2000 stability evaluation and Lysholm function score of knee joint were compared at 6 months after operation. RESULTS: All patients were followed up for 8 to 16 months with an average of (12.3±1.9) months. There were no complications such as incision infection, fracture malunion or non-union, and internal fixation loosening occurred. The avulsion fractures of knee joint were reached to imaging healing standard at 6 months after operation. Operation time and blood loss in open reduction and internal fixation group were (56.4±7.1) min and (63.2±10.2) ml, while (89.9±7.4) min and (27.7±8.7) ml in arthroscopic fixation group, respectively, and had significant difference between two groups (P<0.05). There were no differences in immediate reduction quality (χ2=0.257, P=0.612), knee joint range of motion at 6 months after opertaion (t=0.492, P=0.626), knee joint rear drawer test ( χ2=0.320, P=0.572), IKDC classification of knee joint (χ2=0.127, P=0.938), KT2000 stability evaluation (χ2=0.070, P=0.791), and knee Lysholm function score (t=0.092, P=0.282) between two groups. CONCLUSION: Posterior medial approach with hollow anchoring system fixation and arthroscopic Endobutton with loop plate fixation for the treatment of posterior cruciate ligament tibial occlusion avulsion fracture could achieve satisfactory clinical results, and arthroscopic surgery has less bleeding, but also has a longer learning curve and longer operation time than traditional incision surgery. The surgeon needs to make a choice according to clinical situation of patient and their own surgical inclination.

[Clinical effect of allogeneic peroneal bone marrow support combined with plate fixation for the treatment of Neer type â £ proximal humeral fractures].

OBJECTIVE: To explore clinical effect of allogeneic peroneal bone marrow support combined with plate internal fixation in treating Neer type Ⅳproximal humeral fractures. METHODS: From December 2017 to December 2020,12 patients with Neer type Ⅳ proximal humeral fractures were treated with allogeneic peroneal bone marrow support combined with plate internal fixation,including 7 males and 5 females,aged from 56 to 78 years old;the time from injury to operation ranged from 1 to7 days. Operative time,fracture healing time and complications during follow-up were observed,and clinical efficacy was evaluated by Constant-Murley score at the latest follow-up. RESULTS: All patients were obtained follow up for 20 to 29 months. All patients got bone healing and incisicons were healed at stageⅠ,operative time ranged from 95 to 138 min,blood loss ranged from 210 to 275 ml,fracture healing time ranged from 14 to 18 weeks. Two patients occurred postoperative shoulder stiffness and recovered after 2 weeks of passive exercise. There were no complications such as infection,poor wound healing,and failure (fracture and loosening) of internal fixators occurred. Constant-Murley shoulder function score ranged from 69 to 89 at the latest follow up,2 patients got excellent results,9 good and 1 fair. CONCLUSION: The application of allogeneic fibular bone marrow placement could provide effective support for medial humerus,which is conducive to assisting reduction of fracture end,reducing occurrence of internal fixation failure caused by collapse of humerus head and screw perforation,and significantly improving function of shoulder joint.

Significance of circulating tumor cells detection in tumor diagnosis and monitoring.

To detect circulating tumor cells (CTCs) in the peripheral blood of patients with tumor, and to analyze the significance of CTC detection in tumor diagnosis and monitoring. In the present study, peripheral blood was collected from 125 patients with tumor, and CTCs were isolated and identified. Differences in CTC number and subtype detection were analyzed for different tumor diseases and stages. CTCs were detected in 122 of the 125 patients with tumor, with a positive rate of 97.6%. The number of CTCs increases in patients with vascular metastasis. The number of mesenchymal CTCs increases in patients with lymph node or vascular metastasis. The average ratio of epithelial CTCs in each positive sample decreases in the later stages of cancer compared with the earlier stages, while the average ratio of mesenchymal CTCs increases in the later stages of cancer compared with the earlier stages. The results showed that CTCs with mesenchymal phenotypes are closely related to lymph node or vascular metastasis. CTC detection can help with early diagnosis of tumor diseases. Continuous monitoring of changes in CTCs number and subtypes can assist clinical judgment of tumor disease development status and prognosis.

Hepatitis C virus nonstructural protein 4B induces lipogenesis via the Hippo pathway.

Hepatitis C virus (HCV) infection causes abnormal lipid metabolism in hepatocytes, which leads to hepatic steatosis and even hepatocellular carcinoma. HCV nonstructural protein 4B (NS4B) has been reported to induce lipogenesis, but the underlying mechanism is unclear. In this study, western blots were performed to investigate the effect of NS4B protein levels on key effectors of the Hippo and AKT signaling pathways. Yes-associated protein (YAP) and moesin-ezrin-radixin-like protein (Merlin) are effectors of the Hippo pathway. NS4B downregulated Merlin and phosphorylated YAP (p-YAP) protein expression while increasing the expression of the key AKT pathway proteins p-AKT and NF-κB. By observing the levels of AKT pathway proteins when Merlin was overexpressed or silenced, it was determined that Merlin mediates the AKT pathway. We suggest that HCV NS4B may mediate the AKT signaling pathway by inhibiting the Hippo pathway. Lipid droplets were observed in Huh7.5 cells overexpressing NS4B, and they increased significantly in number when Merlin was silenced. Overexpression of NS4B and Merlin silencing enhanced the expression of sterol regulatory element binding proteins (SREBPs), which have been demonstrated to be key regulatory factors controlling fatty acid synthesis. NS4B and Merlin silencing also enhanced the in vitro proliferative capacity of hepatocellular carcinoma cells. In conclusion, NS4B induces lipogenesis via the effect of the Hippo-YAP pathway on the AKT signaling pathway and thereby plays a significant role in the pathogenesis of HCV-associated diseases.

[Treatment of severe medial tibial bone defect in primary total knee arthroplasty with autogenous bone graft and plate fixation].

OBJECTIVE: To explore the technique of autogenous bone graft combined with plate fixation in total knee arthroplasty(TKA) with severe proximal medial tibial bone defect. METHODS: From March 2012 to October 2018, 21 patients (9 males and 12 females) with severe bone defects in the proximal medial tibia during primary total knee arthroplasty were treated with autogenous structural bone grafting and steel plate fixation, with an age of 61 to 77 years old with an average of (69.6±9.1) years and a course of 64 to 257 months with an average of (73.6±170.7) months. According to Rand classification, there were 13 cases of type Ⅲb and 8 cases of type Ⅳb. Postoperative complications were observed, and knee joint function was evaluated by the Hospital for Special Surgery (HSS) score and SF-36 quality of life score. RESULTS: All 21 patients were followed up for 37 to 64 months with an average of (49.5±13.7) months. The incisions of all patients healed smoothly, and 2 patients developed lower limb intermuscular venous plexus thrombosis after operation. There were no periprosthetic infection, loosening of prosthesis and other complications. The autogenous bone grafts of all patients achieved bony healing during postoperative X-ray follow-up, and the healing time was 8 to 13 months with an average of (10.1±2.3) months. The HSS score of patients increased significantly from 30 to 48 with an average of (53.4±4.2) before operation to 75 to 92 with an average of (81.2±8.4) at the final follow-up (P<0.05). The SF-36 quality of life score of patients after operation was significantly different from that before operation (P<0.05). CONCLUSION: The technique of autogenous bone graft combined with steel plate fixation can achieve satisfactory osseointegration effect in the treatment of severe proximal tibial bone defects in primary knee arthroplasty, with less complications and obvious improvement in knee function.

[Case-control study on carpal canal endoscopy and arthroscopy for the treatment of plantar fasciopathy].

OBJECTIVE: To explore clinical effects of carpal canal endoscopy in treating patients with plantar fasciopathy who failed by conservative treatment. METHODS: From August 2018 to August 2019, 50 patients with plantar fascia were divided into two groups and 25 patients in each group. In carpal canal endoscopy group, included 11 males and 14 females, aged from 39 to 67 years old with an average of(57.7±6.4) years old;carpal canal endoscopy was used to plantar fascia release. In arthroscopy group, included 9 males and 16 females, aged from 41 to 73 years old with an average of (58.1±7.2) years old;conventional 4.0 mm arthroscopy Instruments was used to plantar fascia release. Operation time, hospitalization expense and postoperative complications between two groups were observed and compared. Postoperative visual analogue scale(VAS) and American Orthopedic Foot Ankle Society (AOFAS) score were used to evaluate clinical function. RESULTS: All patients were followed up from 12 to 18 months with an average of (14.3±2.1) months. There were significant differentces in operation time and hospitalization expense between two groups (P<0.05). Surgical incision healed well in carpal canal endoscopy group, and 2 patients delayed union in arthroscopy group, and no difference between two groups (P>0.05). There were no statistical differences in VAS, AOFAS and grading between two groups at 12 months after operation(P>0.05). CONCLUSION: The outcome of carpal canal endoscopy and arthroscopy has similar effects in treating plantar fascia. While carpal canal endoscopy has advantages of need not perfusion during opertaion, protect soft tissue well, less operation time, and lower cost.

[Value of quantitative CT in vertebroplasty for osteoporotic fracture combined with scoliosis].

OBJECTIVE: To investigate the value of lumbar quantitative CT (QCT) in vertebroplasty for osteoporotic fracture combined with scoliosis. METHODS: The clinical data of 60 patients with osteoporotic fractures combined with different degrees of scoliosis treated by vertebroplasty from December 2017 to December 2019 were retrospectively analyzed. There were 18 males and 42 females, aged from 65 to 81 (72.63±3.34)years old. All patients were received QCT examination before surgery. According to the QCT value, the patients were divided into osteopenia group(QCT>80 g/L, 10 cases, 12 vertebrae), osteoporosis group(QCT 40-80 g/L, 35 cases, 48 vertebrae) and severe osteoporosis group(QCT<40 g/L, 15 cases, 22 vertebrae). The dispersion and leakage of bone cement in the injured vertebrae of patients with different degrees of QCT value were observed, and the QCT value in the selection of puncture point, correction of Cobb angle and recovery of vertebral height were analyzed in the patients. RESULTS: Among 60 cases of 82 vertebrae, 41 cases of 55 vertebrae were punctured by concave unilateral puncture, according for 67.07%. Among them, there were 2 cases with 2 vertebrae in osteopenia group, 26 cases with 35 vertebrae in osteoporosis group, and 13 cases with 18 vertebrae in severe osteoporosis group. There was significant difference in the number of cases with unilateral or bilateral puncture among the three groups (χ2=13.699, P=0.001); there was no significant difference in the number of cases with bone cement leakage among the three groups (χ2=1.403, P=0.496). The Cobb angle of scoliosis was significantly differentbetween preoperative and postoperative follow-up(P<0.05);the height of injured vertebral body was significantly different between preoperative and postoperative follow-up (P<0.05). CONCLUSION: For patients with osteoporotic fracture combined with scoliosis undergoing vertebroplasty, the severity of osteoporosis should be determined according to lumbar QCT detection, and the concave side of scoliosis should be selected for puncture, which is conducive to improving scoliosis, restoring spinal stability and improving surgical safety.

Performance and Transcriptional Response of the Green Peach Aphid Myzus persicae to the Restriction of Dietary Amino Acids.

Free amino acids in the phloem sap are the dominant nitrogen source for aphids, but their availability is usually poor. Although some studies have explored the effect of dietary amino acid restriction on aphid performance, little is known about the molecular basis of these effects. Here, we examined the performance and transcriptome of the green peach aphid, Myzus persicae, fed a standard diet (Control diet) or a diet containing 50% of the total amino acids of the Control diet (Half diet). Aphid weight and fecundity were significantly reduced in the Half diet group. Transcriptomic analysis showed that a total of 1460 genes were differentially expressed between the groups were fed on the two diets, which many of them were associated with nutrient and energy metabolism. When feeding on the Half diet, aphids upregulated genes associated with the amino acid biosynthetic pathway (predominantly amino acid biosynthesis genes and some amino acid transporter genes) as well as the cysteine and serine protease genes. Furthermore, these aphids displayed increased expression of genes associated with glycolysis, which could generate intermediates for de novo amino acid biosynthesis. Consistent with this, elevated glucose levels were observed in aphids in the Half diet group. Additionally, the expression levels of several genes associated with hormonal signaling pathway were altered. Several genes related to juvenile hormone and insulin-like peptide (ILP) signaling were downregulated, including Krüppel homolog 1 (Kr-h1) and insulin-like peptide 5 (Ilp5), respectively. In contrast, several genes related to ecdysone signaling were upregulated including broad-complex core protein (Br-c) and shade (Shd). Despite their poor performances, M. persicae adapted to dietary restriction of amino acids, through upregulation of genes involved in amino acid biosynthesis, glycolysis, and protein degradation, as well as by altering the expression level of genes involved in hormone signaling pathways.

The L-DOPA/Dopamine Pathway Transgenerationally Regulates Cuticular Melanization in the Pea Aphid Acyrthosiphon pisum.

Maternal phenotypic regulations between different generations of aphid species help aphids to adapt to environmental challenges. The pea aphid Acyrthosiphon pisum has been used as a biological model for studies on phenotypic regulation for adaptation, and its alternative phenotypes are typically and physiologically based on maternal effects. We have observed an artificially induced and host-related maternal effect that may be a new aspect to consider in maternal regulation studies using A. pisum. Marked phenotypic changes in the cuticular melanization of daughter A. pisum were detected via tyrosine hydroxylase knockdown in the mothers during their period of host plants alternations. This phenotypic change was found to be both remarkable and repeatable. We performed several studies to understand its regulation and concluded that it may be controlled via the dopamine pathway. The downregulation and phenotypes observed were verified and described in detail. Additionally, based on histological and immunofluorescence analyses, the phenotypic changes caused by cuticular dysplasia were physiologically detected. Furthermore, we found that this abnormal development could not be reversed after birth. Transcriptome sequencing confirmed that this abnormal development represents a systemic developmental failure with numerous transcriptional changes, and chemical interventions suggested that transgenerational signals were not transferred through the nervous system. Our data show that transgenerational regulation (maternal effect) was responsible for the melanization failure. The developmental signals were received by the embryos from the mother aphids and were retained after birth. APTH RNAi disrupted the phenotypic determination process. We demonstrate that non-neuronal dopamine regulation plays a crucial role in the transgenerational phenotypic regulation of A. pisum. These results enhance our understanding of phenotyping via maternal regulation in aphids.

[Analysis of the treatment of posterolateral tibial plateau fracture with three different methods].

OBJECTIVE: To analyze and compare the efficacy of surgical approaches and fixations of anterolateral approach, lateral approach and posterolateral approach in treating posterolateral tibial plateau fracture. METHODS: A retrospective study of 44 cases from May 2010 to July 2014 were enrolled, of which there were 21 males and 23 females, and the mean age was 42.5 years old (ranged, 26 to 61 years). All the cases were divided into 3 groups according to the surgical approach, group A was anterolateral approach (19 cases), group B was lateral approach (15 cases), group C was posterolateral approach (10 cases). Operative time and bleeding volum were compared and the knee function was observed. RESULTS: The mean operative time of group A was (91.3±10.4) min, and the bleeding volum of which was (175.3±20.3) ml. The mean operative time of group B was(86.6±9.2) min, and the bleeding volum of which was(155.8±18.2) ml. The mean operative time of group C was (109.5±10.8) min, and the bleeding volum of which was(235.9±29.1) ml. There were significant differences in operative time and bleeding volum between group C and the other two groups(P<0.05). The mean follow-up time was 14.9 months (ranged, 10 to 35 months), and the HSS score of last follow-up was 89.6±7.5 (group A), 90.2±6.4(group B), 88.9±5.1 (group C). There were no significant differences in groups(P>0.05). CONCLUSIONS: The operative time of posterolateral approach was longer than anterolateral approach or lateral approach, as well as the bleeding volum which was higher in posterolateral approach, while no significant difference of the knee function was observed in these 3 different approaches.

Deciphering the Function of Octopaminergic Signaling on Wing Polyphenism of the Pea Aphid Acyrthosiphon pisum.

Aphids exhibit wing polyphenism (winged or wingless) for adaption to predictable or temporally heterogeneous environmental changes; however, the underlying mechanism is still unclear. This morphological change could be stimulated by high aphid density, which in turn could affect octopaminergic signaling in aphids. Octopamine is a neurotransmitter synthesized in insects that can modify their physiological metabolism, locomotion, and other behaviors. We designed experiments to determine whether octopamine functions in wing formation of the pea aphid, Acyrthosiphon pisum (Harris). We determined gene expression of tyramine ß-hydroxylase (TßH), a key enzyme in octopamine synthesis at different developmental stages, in different body parts, and in different densities of aphids. We also used TßH RNAi, octopamine receptor agonists (octopamine and synephrine), and an antagonist (mianserin) to modify octopaminergic signaling. We found that transcription of TßH was related to aphid density, which affected the proportion of winged offspring. By manually modifying the mother's octopaminergic signaling, TßH expression was suppressed, and TßH (enzyme) activity decreased. The proportion of winged offspring was also affected. Our results showed that octopamine could be a link in the wing determination system, as well as environmental stimulation. The RNAi results showed that the decrease of TßH expression increased aphid's reproduction; however, the decrease of TßH expression declined the numbers of winged-offspring producers, but did not affect the proportion of winged nymphs produced by the winged-offspring producer. In conclusion, the decline in the proportion of winged daughters in the next generation was caused by the decline of winged nymph producers.

HCV NS4B targets Scribble for proteasome-mediated degradation to facilitate cell transformation.

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is a multi-transmembrane protein, but little is known about how NS4B contributes to HCV replication and tumorigenesis. Its C-terminal domain (CTD) has been shown to associate with intracellular membrane, and we have previously shown that NS4B CTD contains a class I PDZ-binding motif (PBM). Here, we demonstrated that NS4B PBM interacts with the PDZ-containing tumor suppressor protein, Scribble, using immunofluorescence and co-immunoprecipitation assays, and this interaction requires at least three contiguous PDZ domains of Scribble. In addition, NS4B PBM specifically induced Scribble degradation by activating the proteasome-ubiquitin pathway. Similar Scribble degradation was also observed in HCV-infected cells, suggesting NS4B could work in the context of HCV. Finally, NS4B PBM mutants showed reduced colony formation capacity compared with its wild-type counterpart, indicating that NS4B PBM plays important roles in NS4B-mediated cell transformation. Altogether, we provide a mechanism by which NS4B induces cell transformation through its PBM, which specifically interacts with the PDZ domains of Scribble and targets Scribble for degradation.

Anthocyanin Accumulation, Antioxidant Ability and Stability, and a Transcriptional Analysis of Anthocyanin Biosynthesis in Purple Heading Chinese Cabbage (Brassica rapa L. ssp. pekinensis).

Heading Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a significant dietary vegetable for its edible heading leaves in Asia countries. The new purple anthocyanin-rich pure line (11S91) was successfully bred, and the anthocyanins were mainly distributed in 2-3 cell layers beneath the leaf epidermis, whereas siliques and stems accumulated only a cell layer of anthocyanins. The anthocyanins of 11S91 were more stable at pHs below 3.0 and temperatures below 45 °C. The total antioxidant ability was highly positive correlated with the anthocyanin content in 11S91. Thirty-two anthocyanins were separated and identified, and 70% of them were glycosylated and acylated cyanidins. The four major anthocyanins present were cyanidin-3-sophoroside(p-coumaroyl)-5-glucoside(malonyl), cyanidin-3-sophoroside(ferulyl)-5-glucoside(malonyl), cyanidin-3-sophoroside(sinapyl-p-coumaroyl)-5-glucoside(malonyl), and cyanidin-3-sophoroside-(sinapyl-ferulyl)-5-glucoside(malonyl). According to the expression of biosynthetic genes and the component profile of anthocyanins in 11S91 and its parents, regulatory genes BrMYB2 and BrTT8 probably activate the anthocyanin biosynthesis but other factors may govern the primary anthocyanins and the distribution.

Cross-linked polyethylenimine-tripolyphosphate nanoparticles for gene delivery.

The high transfection efficiency of polyethylenimine (PEI) makes it an attractive potential nonviral genetic vector for gene delivery and therapy. However, the highly positive charge of PEI leads to cytotoxicity and limits its application. To reduce the cytotoxicity of PEI, we prepared anion-enriched nanoparticles that combined PEI with tripolyphosphate (TPP). We then characterized the PEI-TPP nanoparticles in terms of size, zeta potential, and Fourier-transform infrared (FTIR) spectra, and assessed their transfection efficiency, cytotoxicity, and ability to resist deoxyribonuclease (DNase) I digestion. The cellular uptake of PEI-TPP with phosphorylated internal ribosome entry site-enhanced green fluorescent protein C1 or FAM (fluorouracil, Adriamycin [doxorubicin] and mitomycin)-labeled small interfering ribonucleic acids (siRNAs) was monitored by fluorescence microscopy and confocal laser microscopy. The efficiency of transfected delivery of plasmid deoxyribonucleic acid (DNA) and siRNA in vitro was 1.11- to 4.20-fold higher with the PEI-TPP particles (7.6% cross-linked) than with the PEI, at all N:P ratios (nitrogen in PEI to phosphorus in DNA) tested. The cell viability of different cell lines was more than 90% at the chosen N:P ratios of PEI-TPP/DNA complexes. Moreover, PEI-TPP nanoparticles resisted digestion by DNase I for more than 2 hours. The time-dependent absorption experiment showed that 7.6% of cross-linked PEI-TPP particles were internalized by 293T cells within 1 hour. In summary, PEI-TPP nanoparticles effectively transfected cells while conferring little or no toxicity, and thus have potential application in gene delivery.

Effect of CD95 on inflammatory response in rheumatoid arthritis fibroblast-like synoviocytes.

OBJECTIVE: Many CD95-expressing cells don't always undergo apoptosis after stimulation with CD95 ligation. The purpose of this paper is to investigate the role of expression of CD95 (Fas/Apo1) on inflammatory response in fibroblast-like synoviocytes (FLS) obtained from rheumatoid arthritis (RA) and to evaluate the role of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB or Akt) pathways within this process. METHODS: The expression levels of CD95 were monitored by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR). Apoptotic cells were detected by in situ apoptosis detection (TUNEL) assay. The RA-FLS were treated with agonistic anti-CD95 antibody or CD95 siRNA. Then the proliferation was detected by CCK-8, and mRNA level of inflammatory cytokines was detected by RT-PCR. After the RA-FLS were treated with agonistic anti-CD95 antibody, the total Akt and pAkt protein expression was analyzed by Western blot, and the changes mentioned above were observed while pre-incubated with the PI3K inhibitor LY294002. RESULTS: A significant increase of CD95 antigen was found in RA compared with osteoarthritis (OA) samples, while apoptosis in RA synovial tissue was not obvious. Low concentrations of agonistic anti-CD95 antibody could promote RA-FLS growth and interleukin-6 (IL-6) mRNA expression, while high concentrations could induce apoptosis. And both of these phenomena could be inhibited by CD95 siRNA. Agonistic anti-CD95 antibody could stimulate the expression of pAkt, and PI3K specific inhibitor LY294002 could induce opposite changes. CONCLUSION: Stimulation of CD95 could promote RA-FLS proliferation and inflammation, and activation of the PI3K/Akt signaling pathway might be the possible mechanism.

A potential skin substitute constructed with hEGF gene modified HaCaT cells for treatment of burn wounds in a rat model.

This study aimed to investigate the feasibility of using an immortal keratinocyte cell line, HaCaT cells, to effectively deliver epidermal growth factor (EGF) in a skin substitute to treat burn wounds. The skin equivalent was constructed with human EGF (hEGF) gene modified HaCaT cells obtained through stable gene transfection; these were applied to full thickness burn wounds in a rat model. The results showed that the hEGF gene modified HaCaT cells produced more than 390ng/l of bioactive hEGF in the culture supernatant. K19 and integrin-ß1 as keratinocyte differentiation markers were elevated in the hEGF gene modified HaCaT cells which were shown to be non-tumorigenic. The skin equivalent constructed with hEGF gene modified HaCaT cells demonstrated improved epidermal morphogenesis with a thick and compact epidermis. Wound healing was accelerated noticeably when applied with this skin substitute seeded with hEGF gene modified HaCaT cells in vivo. The results suggest that HaCaT cells modified with hEGF gene might be promising seed cells for construction of genetically modified skin substitute which can effectively secrete hEGF to accelerate wound repair and regeneration.

[Biological effects of paracrine from insulin stimulated adipose-derived stem cells (ADSC) on human vascular endothelial cells].

OBJECTIVE: To study the biological effects of the paracrine from ADSC after being stimulated by insulin on vascular endothelial cells. METHODS: (1) ADSC was isolated from human adipose tissue and cultured in vitro. The third generation cells were collected and divided into insulin group (I, cultured with serum-free DMEM containing 1 x 10(-7) mol/L insulin) and control group (C, cultured with serum-free DMEM) according to the random number table, with 6 slots in each group. Three days later, ADSC culture medium (ADSC-CM) was collected for determination of levels of vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) by ELISA. (2) Human umbilical vein endothelial cells (HUVEC) were cultured to the third generation, and they were cultured with special nutrient solution and divided into ADSC-CM with insulin stimulation group (AI), ADSC-CM without insulin stimulation group (AC), insulin group (I, with same concentration as above), blank control group (BC) according to the random number table. Three days later, proliferation of HUVEC was determined with MTT method (with expression of absorbance value). Another two samples of HUVEC were respectively divided into 4 groups as above for determination of apoptosis rate with Annexin V/FITC double-staining 12 hours after culture, and HUVEC migration with scratch adhesion test at post scratch hour (PSH) 12, 24, 36, 48. Data were processed with t test. RESULTS: (1) Compared with those in C group [(287 +/- 47), (577 +/- 84) pg/mL, respectively], the secretion levels of VEGF and HGF in I group [(643 +/- 64), (930 +/- 68) pg/mL, respectively] were significantly increased (with t value respectively 18.869, 18.475, P values all below 0.05). (2) The absorbance value of HUVEC in AI and AC groups was 0.847 +/- 0.042, 0.798 +/- 0.022, respectively, which were higher than that in I and BC groups [0.665 +/- 0.028 (with t value respectively 4.579, 3.732), 0.674 +/- 0.031 (with t value respectively 3.761, 4.073), P values all below 0.01], and that in AI group was higher than that in AC group (t = 2.576, P < 0.05). The apoptosis rates of HUVEC in AI and AC groups [(5.8 +/- 1.9)%, (9.0 +/- 2.0)%, respectively] were obviously lower as compared with that in I and BC groups [(30.4 +/- 6.0)% (with t value respectively 12.891, 10.417), (31.4 +/- 7.4)% (with t value respectively 11.474, 9.783), P values all below 0.05 ], and that in AC group was higher than that in AI group (t = 8.548, P < 0.05). The distance of migration of HUVEC in AI and AC groups were greater than that in I and BC groups at PSH 36, 48, and that in AI group was greater as compared with that in AC group (with t value respectively 4.076, 4.573, P values all below 0.05). CONCLUSIONS: Paracrine from ADSC after being stimulated by insulin can promote proliferation and migration of HUVEC, and suppress its apoptosis, and it is beneficial for tissue vascularization.

[Experimental study on culture method of human umbilical vein endothelial cells].

OBJECTIVE: To establish an efficient and stable culture method of human umbilical vein endothelial cells (HUVECs) in vitro so as to provide good source of seed cells for tissue engineered vascular grafts and for preclinical research. METHODS: The umbilical cords were harvested from full-term normal delivered neonates, which were perfused with 0.1% collagenase II by self-made needle and were digested at 37 degrees C and 5% CO2 humidified incubator. The HUVECs were cultured in endothelial culture medium (ECM) containing 5% fetal bovine serum (FBS) and 1% endothelial cell growth factor (ECGS). HE staining of the umbilical cords before and after digestion was used to observe the detachment of HUVECs, flow cytometry to detect the purity of primary HUVECs, and inverted phase contrast microscope to observe the morphology of the cultured HUVECs. The growth of the 3rd passage cells was measured by MTT assay; immunocytochemical technique and matrigel-based capillary-like tube formation assay were carried out to identify the function of HUVECs. RESULTS: After digestion of 0.1% collagenase II, marked HUVECs detachment was observed with complete digestion. The purity of the HUVECs was 99.56% by digestion of 0.1% collagenase II at 37 degrees C and 5% CO2 humidified incubator for 15 minutes. Primary HUVECs showed a cobblestone or pitching stone-like appearance in vitro, forming a confluent monolayer cells after 2-3 days of culture. MTT assay demonstrated that HUVECs showed the fastest growth speed at 3 to 4 days, and showed growth of cell fusion at about 5 days. Immunocytochemistry showed that HUVECs highly expressed endothelial marker factor VIII. Matrigel based capillary-like tube formation assay showed that it could form endothelial-like tube structures after 24 hours of culture. CONCLUSION: Using improved method and ECM could obtain high quantity and high quality primary HUVECs, which might be a kind of promising seed cells for tissue engineering and preclinical research.

[Cytobiological effect of adipose-derived stem cells treated with insulin on HaCaT cells].

OBJECTIVE: To isolate and culture adipose-derived stem cells (ADSCs), and to study the effects of the conditioned medium of ADSCs (ADSC-CM) treated with insulin on HaCaT cells. METHODS: ADSCs were isolated from adipose tissue donated by the patient receiving abdominal surgery and were cultured. The concentration of ADSCs at passage 3 was adjusted to 5 x 10(4) cells/mL. The cells were divided into 2 groups: group A in which the cells were incubated in 1 x 10(-7) mol/L insulin for 3 days, and group B in which the cells were not treated with insulin. ADSC-CM in each group was collected 3 days after culture, then levels of VEGF and hepatocyte growth factor (HGF). HaCaT cells were cultured and the cells at passage 4 were divided into 4 groups: group A1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group A; group B1, 0.5 mL 2% FBS and 0.5 mL ADSC-CM from group B; group C1, 1 mL 2% FBS of 1 x 10(-7) mol/L insulin; group D1, 1 mL 2% FBS. Proliferation of HaCaT cells was detected by MTT method 3 days after culture, apoptosis rate of HaCaT cells was measured by Annexin V-FITC double staining 12 hours after culture, and the migration ability was measured by in vitro wound-healing assay 0, 12, 24, 36 and 48 hours after culture. RESULTS: The level of VEGF in groups A and B was (643.28 +/- 63.57) and (286.52 +/- 46.68) pg/mL, respectively, and the level of HGF in groups A and B was (929.95 +/- 67.52) and (576.61 +/- 84.29) pg/mL, respectively, suggesting differences were significant between two groups (P < 0.05). Cell proliferation detection showed the absorbance value of HaCaT cells in group A1, B1, C1 and D1 was 0.881 +/- 0.039, 0.804 +/- 0.041, 0.663 +/- 0.027 and 0.652 +/- 0.042, respectively, suggesting there was significant difference between groups A1 and B1 and groups C1 and D1 (P < 0.01), group A1 was significantly higher than group B1 (P < 0.05). The apoptosis rate of HaCaT cells in groups A1, B1, C1 and D1 was 5.23% +/- 1.98%, 8.82% +/- 2.59%, 31.70% +/- 8.85% and 29.60% +/- 8.41%, respectively, indicating there was significant difference between groups A1 and B1 and groups C1 and D1 (P < 0.05), group B1 was significantly higher than group A1 (P < 0.05). The migration distance of HaCaT cells in groups A1, B1, C1 and D1 at 36 hours was (0.184 6 +/- 0.019 2), (0.159 8 +/- 0.029 4), (0.059 2 +/- 0.017 6) and (0.058 2 +/- 0.012 3) mm, respectively, whereas at 48 hours, it was (0.231 8 +/- 0.1740), (0.205 1 +/- 0.012 1), (0.079 2 +/- 0.008 1) and (0.078 4 +/- 0.011 7) mm, respectively, suggesting there were significant differences between groups A1 and B1 and groups C1 and D1 at 36 and 48 hours (P < 0.01), group A1 was significantly higher than group B1 (P < 0.05) at 36 and 48 hours, no significant difference was evident at other time points (P > 0.05). CONCLUSION: ADSCs treated with insulin can significantly promote the proliferation and the migration of HaCaT cells and inhibit their apoptosis.

Porcine reproductive and respiratory syndrome viruses predominant in southeastern China from 2004 to 2007 were from a common source and underwent further divergence.

The full-length glycoprotein 5 (GP5) gene and a partial nonstructural protein 2 (NSP2) gene fragment of 46 porcine reproductive and respiratory syndrome viruses (PRRSV) from pig farms in southeastern China between 2004 and 2007 were sequenced for phylogenetic analysis. All of the PRRSV isolates in this study were of the North American type, and the majority of them were clustered in subgroup II and had 84.1-89.1% amino acid sequence identity to those of subgroup I including the North American strain VR-2332. Three variable regions containing epitopes A and B in the N-terminal region were identified and found to be under positive selection. Several additional mutations, which were also located in the variable regions, were seen in isolates from the years 2006 and 2007 in subgroup II, as compared with those of earlier years (2004-2005) in the same group. Further analysis revealed that the majority of the subgroup II PRRSV isolates prevalent in the region since 2004 had thirteen mutation sites that distinguished them from subgroup I strains, indicating a possible introduction of a certain strain from the same source in the region or elsewhere well before 2004. A 29-aa deletion in the NSP2 fragment was found in PRRSV isolates as early as in 2005, one year earlier than the virulent PRRSV with the same deletion became dominant in China. Taken together, this study shows that subgroup II PRRSV strains with a partial deletion of nsp2 are currently prevailing in southeastern China.