Objective To observe angiogenic effect of platelet-released growth factors (PRGF) from platelet-rich plasma (PRP) on mierovessel formation at early stage after anterior crueiate ligament (ACL) reconstruction by freeze-dried Achilles tendon. Methods The study involved 14 rabbits, of which 12 rabbits were used as experiment group and the other 2 as control group. In the experiment group, after two sides of rabbit ACL were removed, freeze-dried Achilles tendon treated by PRGF was transplanted into random one side of the knee to substitute the original ACL (PRGF group), while the other side was transplanted with freeze-dried Achilles tendon treated only by normal saline (NS group). Only one side of the knee was removed in the control group. The grafts were observed by HE and immuno- histeehemical staining 2, 4 and 6 weeks after operation. Microvessel density (MVD) was measured by Weidner method. Results Compared with NS group, MVD in PRGF group was significant higher at 2,4 and 6 weeks after operation (P<0.05). MVD of NS group at 2,4 and 6 weeks after operation was 2.52±0.45, 3.41±0.44 and 2.57±0.51 respectively, but that of PRGF group at 2,4 and 6 weeks af- ter operation was 3.56±0.81,4.91±0.46 and 3.01±0.75 respectively (P<0.05). The time of neo- vascular formation and the depth of vascular penetration into the grafts of the PRGF group were superior to those of NS group. Conclusion PRGF can significantly promote microvessel formation at early stage after ACL reconstruction with freeze-dried Achilles tendon.

Objective To observe the effect of platelet-rich plasma(PRP)gel on tendon-bone healing following tendon allograft reconstruction of anterior cruciate ligament(ACL).Methods Bilateral ACL reconstructions using Achilles tendon allografts were performed in 24 New Zealand white rabbits matured skeletally.One knee joint was pretreated with the allograft PRP gel(served as experimental group),while the contralateral knee joint was free from treatment with PRP(served as control group).The reconstructions were assessed histologically,immunohistochemically and biomechanically at 2,6 and 12 weeks.Results At 2 and 6 weeks,Burak scores of experimental group were higher than control group.At 12 weeks,the grafts showed a mature zone of fibrocartilage in experimental group but mature scar tissues on the tendon-bone surface.Immunohistochemistry demonstrated early higher expression of VEGF in experimental group than control group and continually higher expression of TGF-β1 in experimental than control group.In contrast,the grafts of the controls group revealed the development of mature scar tissue resembling Sharpey fibers spanning the tendon-bone interface.At 2 and 6 weeks,the biomechanical analysis revealed the limit load of(15.3±2.9)N and(33.2±6.9)N respectively in experimental group,which were significantly higher than(7.9±1.4)N and(23.7±4.9)N in control group (P ＜ 0.05).Conclusion Application of PRP is the potential means to enhance the earlier healing of the allograft tendon-bone.

Objective:To investigate the effects of riboflavin-ultraviolet A scleral collagen cross-linking on the retina under different irradiation time, and to determine the safe irradiation time.Methods:Sixty healthy New Zealand white rabbits were randomly divided into control group (0 minute group), 10 minutes group, 20 minutes group, 30 minutes group and 40 minutes irradiation group according to the irradiation time, with 12 rabbits in each group.The left eye was irradiated with riboflavin-ultraviolet A scleral collagen (370 nm, 10 mW/cm 2). The histopathological change of retina was observed by light microscope and transmission electron microscope and compared among different groups.The concentration of MDA and the activities of SOD, CAT and GSH-Px in retinal tissue were detected by corresponding kits.The expression levels of SOD and CAT proteins in retinal tissue were detected by Western blot method.The study protocol was approved by the Binzhou Medical University Laboratory Animal Ethical Committee (No.2017-80). The use and care of animals complied with the statement of ARVO and the Regulation on the Management of Laboratory Animal Quality of China. Results:Under the light microscope, the structure of the retinas in the control group was orderly arranged.Under the transmission electron microscope, the lamellar structure in the inner segment and the mitochondrial structure in the outer segment of the photoreceptor cells were intact, and the mitochondrial ridge was continuous in the control group.There was no obvious difference in retinal morphology between the 10 minutes irradiation group and the control group under both the light microscope and the transmission electron microscope, and the retinal damage became more severe with the prolongation of irradiation time.The concentration of MDA in the retina of each group was elevated gradually with the increase of irradiation time, and the difference was statistically significant ( F=65.25, P<0.05). The concentration of MDA was (11.31±1.84), (14.94±1.04)and (18.25±1.42)nmol/mgprot in the 20 minutes, 30 minutes and 40 minutes irradiation groups respectively, which were significantly higher than (1.13±0.02)nmol/mgprot in the control group (all at P<0.05). The MDA concentration in 20 minutes, 30 minutes and 40 minutes irradiation groups was increased successively, showing statistically significant differences (all at P<0.05). With the prolongation of irradiation time, the activities of SOD, CAT and GSH-Px as well as the expression levels of SOD and CAT proteins were significantly decreased gradually ( F=44.09, 34.18, 35.60, 115.75, 78.86; all at P<0.05). The differences between the control group and 20 minutes, 30 minutes, 40 minutes irradiation groups, and the differences among 20 minutes, 30 minutes, 40 minutes irradiation groups were statistically significant (all at P<0.05). Conclusions:Riboflavin-ultraviolet A 10 mW/cm 2 scleral collagen cross-linking irradiation for 10 minutes is safe.Excessive irradiation time can cause damage to the retina of rabits.

To analyze a case of severe avian influenza A (H5N6) virus infection resulting in severe pneumonia and acute respiratory distress syndrome (ARDS) was admitted to Guilin Municipal Hospital of Traditional Chinese Medicine on July 6, 2023. The clinical data and treatment of this patient were analyzed retrospectively. The initial clinical manifestations of the patient were fever, cough, and expectoration, and the antigen test for influenza A virus was positive. Chest CT showed: double lung texture increased and thickened, and multiple patchy high-density shadows with air-containing bronchial shadows were found in the left lung, especially in the left upper lobe; a few patchy increased-density shadows were also seen in the lower lobe of the right lung, along with left-sided pleural effusion. Metagenomic next-generation metagenomic sequencing (mNGS) of bronchoalveolar lavage fluid was performed to identify the pathogen as influenza A virus H5N6. On the 4th day of admission, the patient's condition rapidly progressed to ARDS, which could not be improved by high-flow oxygen therapy, mechanical ventilation, and prone position ventilation. Subsequently, with the assistance of veno-venous extracorporeal membrane oxygenation (VV-ECMO), the patient's lung function gradually improved. Extracorporeal membrane oxygenation（ECMO） was withdrawn after 25 days, and the patient recovered and was discharged after a hospital stay of 41 days. Patients with severe avian influenza A (H5N6) usually have critical illness and rapid progression, often rapidly progressing to ARDS. When conventional mechanical ventilation cannot correct hypoxemia, VV-ECMO auxiliary treatment should be administered as early as possible. In addition, mNGS can help to quickly identify the diagnosis and differential diagnosis of avian influenza A (H5N6) in the early stage of the disease, particularly suitable for the diagnosis of severe and emergency infections.

Objective:To investigate the effects and mechanisms of allogeneic epidermal stem cells (ESCs) on the survival of allogeneic full-thickness skin grafts in nude mice with full-thickness skin defect wounds.Methods:Experimental research methods were applied. Primary ESCs that appeared paving stone-like after being cultured for 7 d were obtained by enzymatic digestion method from one 4-week-old male BALB/c-NU nude mouse (the same strain, age, and sex below). The cells of third passage were identified by flow cytometry to positively express ESC marker CD44 and negatively express CD45, meanwhile, the positive expression of ESC markers of p63 and integrin 6α, and negative expression of CD71 were identified by immunofluorescence method. The ESCs of third passage in the logarithmic growth phase were used for the following experiments. Twenty-six nude mice were equally divided into phosphate buffered saline (PBS) group and ESCs group according to the random number table. A full-thickness skin defect wound was made on the back of each nude mouse, and then the wounds of the two groups were sprayed with equal volumes of PBS and ESCs, respectively. The wounds were transplanted with full-thickness skin grafts cut from the backs of 4 other nude mice. Each ten nude mice from the two groups were selected, the wound healing and skin survival on post surgery day (PSD) 0 (immediately), 3, 7, 14, and 21 were observed, and the survival ratio and shrinkage rate of skin grafts on PSD 3, 7, 14, and 21 were calculated (the number of sample was the number of surviving skin grafts at each time point); the blood perfusion in the skin grafts on PSD 3, 7, and 14 was detected by the laser speckle blood flow imager, and the blood flow ratio of nude mice skin grafts in ESCs group to PBS group at each time point was calculated (the number of sample was the pair number of surviving skin grafts in group pairing at each time point). The skin graft tissue of each 3 nude mice remained in the two groups were collected on PSD 7, and the mRNA expressions and protein expressions of tumor necrosis factor α (TNF-α), interleukin 8 (IL-8), IL-10, type Ⅰ collagen, type Ⅲ collagen, and matrix metalloproteinase 9 (MMP-9) in the tissue were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction and Western blotting, respectively. Data were statistically analyzed with Log-rank test, analysis of variance for repeated measurement, one-way analysis of variance, independent sample t test, and Bonferroni correction. Results:Taking the condition on PSD 0 as a reference, the wounds of nude mice in the two groups healed gradually on PSD 3, 7, 14, and 21, and the shrinkage of skin grafts was gradually obvious. Among them, the shrinkage healing of wound of nude mice in PBS group was more significant than that in ESCs group. On PSD 3, the skin graft of 1 nude mouse failed in ESCs group, while the skin graft of 3 nude mice failed in PBS group. On PSD 7, the skin graft of another nude mouse failed in PBS group. The survival ratio of skin grafts of nude mice in the two groups was similar on PSD 3, 7, 14, and 21 ( P>0.05). On PSD 3, 7, 14, and 21, the shrinkage rates of skin grafts of nude mice in ESCs group were (9.2±0.4)%, (19.7±1.2)%, (53.6±3.5)%, and (62.2±5.1)%, respectively, which was significantly lower than (11.0±0.9)%, (47.8±2.8)%, (86.1±7.1)%, and (89.7±9.0)% in PBS group ( t=5.719, 26.650, 11.940, 7.617, P<0.01). On PSD 3, 7, and 14, blood perfusion signals were observed in the skin grafts of nude mice in the two groups. The average blood perfusion ratios of the skin grafts of nude mice in ESCs group to PBS group were greater than 1, and there was no statistically significant difference in the overall comparison of 3 time points ( P>0.05). On PSD 7, compared with those of PBS group, the mRNA and protein expressions of TNF-α, IL-8, type Ⅰ collagen, and type Ⅲ collagen in the skin graft tissue of nude mice in ESCs group were significantly reduced, while the mRNA and protein expressions of IL-10 and MMP-9 in the skin graft tissue of nude mice in ESCs group were significantly increased (in mRNA comparison, t=2.823, 2.934, 2.845, 2.860, 3.877, 2.916, P<0.05). Conclusions:Allogeneic ESCs can reduce the shrinkage of allogeneic full-thickness skin grafts transplanted on full-thickness skin defect wounds in nude mice, promote the formation of new blood vessels between the skin graft and the wound, reduce inflammation and collagen protein expression, and promote the expression of MMP-9, thus improving the survival quality of skin grafts.

Objective: To study the relationship between the development of hepatocellular carcinoma(HCC) and HBV gene characteristics among the HCC patients with hepatitis B virus (HBV) infection. Methods: Some acute and chronic hepatitis B patients were collected as control group and HBV associated HCC patients as HCC group. Serum samples of subjects were tested for HBV serological markers. HBV DNA of those samples had been extracted and nested PCR was used to amplify the sequence of HBV DNA. Furthermore, MEGA 6.0 and Bioedit softwares were used to made phylogenetic trees and analyze the gene mutations. Results: The sequences of S region and BCP/Precore region of HBV were amplified from 86 samples in study group and 39 samples in control group. The prevalence of PreS deletion, A1762T and A1762T/G1764A in HCC group were 39.53%, 74.42% and 72.09% respectively, and in control group were 20.51%, 53.85% and 53.85% respectively. The statistical differences of them were significant. The prevalence of A1762T and A1762T/G1764A in ≥ 50 years group were higher than that of < 50 years group. The prevalence of A1762T, G1764A and A1762T/G1764A of subjects who infected genotype C were higher than those infected genotype B. On the contrary, the prevalence of G1896A of subjects who infected genotype C were lower than that of genotype B. It was found that ≥ 50 years, genotype C and G1896A mutation were independently associated with HCC. The risk for suffer from HCC of ≥50 years group, genotype C group and G1896A group were 9.349, 28.875 and 7.648 times compared with < 50 years group genotype B group and without G1896A mutation group, respectively. Conclusions The population of ≥50 years or genotype C had a higher prevalence of A1762T, A1762T/G1764A, ≥50years、genotype C、G1896A were independently associated with HCC, as compared with the subjects of the control group.