This study aimed to obtain a recombinant human-source collagen for industrialization. First, based on the Gly-X-Y sequence of human type I collagen, we optimized the hydrophilic Gly-X-Y collagen peptide, designed the human collagen amino acid sequence and the corresponding nucleotide sequence. Next, the expression vector pPIC9K-COL was constructed via endonuclease digestion technology. We obtained an engineering strain of human-source collagen by electrotransforming Pichia pastoris, and then it was fermented, purified and identified. As a result, the expression level reached 4.5 g/L and the purity was over 95%. After amino acid N-terminal sequencing, molecular weight analysis, amino acid analysis and collagenase degradation test, we confirmed that the obtained collagen was consistent with designed primary structure of human-source collagen. After freeze-drying, we analyzed the collagen by scanning electron microscope and cell cytotoxicity, confirming that the collagen has porous fiber reticular structure and superior cytocompatibility. This indicates that human-source collagen has potential to be applied as biomedical material. In conclusion, we successfully obtained the expected human-source collagen and laid a foundation to its further application.
Amino Acid Sequence ; Biocompatible Materials ; Collagen ; analysis ; Freeze Drying ; Humans ; Pichia ; Recombinant Proteins

BACKGROUND: Temporal cortex is related to human and animal's learning and memory capability, it not only accepts the casting fibers of auditory sense, but also closely connects with many brain areas where are related to spatial cognition.OBJECTIVE: To observe the improving effect of thrombolytic capsule on spatial cognitive capability of rats with lateral temporal cortical infarction DESIGN: Completely randomized controlled study SETTING: Department of Neurology, the 175th Hospital of Chinese PLA;Department of Neurology, General Hospital of Chinese PLA; Institute of Psychology of Chinese Academy of Science MATERIALS: This experiment was carried out at Brain Function Laboratory of Institute of Psychology of Chinese Academy of Science from August 1998 to February 1999, 28 SD rats were randomly divided into 3 groups,namely brain stroke group with 10 rats, treatment group with 10 rats and sham operation group with 8 rats.METHODS: Brain infarction model was established on SD rats by using three-dimensional directional photochemistry induction. Rats were anaesthetized with hydrochloride and intravenously injected of rose pink solution in dosage of 20 mg/kg from tail vein. Then incision was made on the left temporal scalp to expose temporal bone, cold photosource with the guidance of optical fiber was used for directional illumination for 20 minutes in area of 2 mm2. Twenty minutes later, rats in treatment group were given intraperitoneal injection of thrombolytic capsule physiological saline of 40 g/L in dosage of 200 mg/kg, which replaced by the same volume of physiological saline in brain stroke group. Rats in sham-operation group received the same operative lamination and injected with the same volume of physiological saline from tail vein and intraperitoneal cavity.MAIN OUTCOME MEASURES: Achievement of Morris water labyrinth(edge type scored 1, randomize type of 2, trend type of 3 and straight-line type of 4).age response time: it was shortened in treatment group and sham operation group than brain infarction group [ on 1st day: 58,50,65 s; 2nd day: 24,27,46 s; 3rd day: 14,17,20 s; 4th day: 11,9,15 s, (P ＜ 0.01 )], but it was similar ing strategy grades: on the 1st day, scores were slightly higher in treatment group and sham operation group than infarction group, but were instable (F=1.167, P ＞ 0.05). On the 2nd day, it was higher in sham operation group than brain stroke group[2.9,2.3, (F=5.5 ,P ＜ 0.05)], but it was similar between treatment group and infarction group[2.6,2.3 scores, (F=3.34,P ＞ 0.05)]. on the 3rd and 4th day, it was higher in treatment group than infarction group [ 3.5,2.7 minutes; 3.7,3.3 minutes, (F=8.92, 14, P ＞ 0.05)]and similar between treatment group and sham operation group(P ＞ 0.05).CONCLUSION: Rat response time was reduced by thrombolytic capsule during the early time of cerebral infarction, changing searching tactics gradually from randomized and edge type to tendency type and straight line type, suggesting thrombolytic capsule can improve the post-ischemia cognitive function, raise training record of cerebral infarctional rats.

Objective To explore the construction method and physicochemical properties of disulfide?bonded hyaluronic acid?functionalized sodium?meter probe for hepatocellular carcinoma, and its biological evaluation in vitro and feasibility of MRI. Methods Synthesis of hyaluronic acid?disulfide?bonded?poly ε?caprolactone (HA?SS?PCL) by disulfide?bonded alkynyl?terminated polycaprolacton (alkyne?SS?PCL) and azido?terminated hyaluronic acid (HA?N3) by clicking chemical reaction, then doxorubicin (DOX) and superparamagnetic iron oxide (SPIO) were encapsulated in HA?SS?PCL core by dialysis method.HA?SS?PCL@DOX/SPIO was prepared and its particle size,DOX and SPIO loading rate were measured. With PBS as control group, the safety of HA?SS?PCL on human hepatocellular carcinoma cells HepG2 and normal liver cells LO2 was evaluated by the methylthiazolyl tetrazolium (MTT) assay, and the cytotoxicity of HA?PCL@DOX/SPIO and HA?SS?PCL@DOX/SPIO on human hepatocellular carcinoma cells HepG2 was evaluated. Immunofluorescence and flow cytometry were used to observe the expression of CD44 receptor on the surface of HepG2 cells in HA?PCL@DOX/SPIO and HA?SS?PCL@DOX/SPIO groups. Through in vitro MRI, PBS was used as the control group to observe the changes of T2 signal intensity of HA?PCL@DOX/SPIO and HA?SS?PCL@DOX/SPIO groups when SPIO concentration was 10, 20, 40, 80 μg / ml. One way ANOVA test and t test were used. Results HA?SS?PCL@DOX / SPIO sodium?meter probes were successfully constructed. The particle size of HA?SS?PCL@DOX/SPIO was (126.9±6.3) nm,and they were spherical with uniform size. The loading rates of DOX and SPIO were 61.4% and 58.7%. MTT assay showed that the survival rate of HepG2 and LO2 cells was more than 80% even at 500 μg/ml of HA?SS?PCL, 66.6% in HA?PCL@DOX/SPIO group and 55.2% in HA?SS?PCL@DOX/SPIO group. Immunofluorescence and flow cytometry showed that HA?PCL@DOX/SPIO and HA?SS?PCL@DOX/SPIO groups all have strong fluorescence, and the latter has stronger fluorescence intensity the former fluorescence intensity was 139.70±8.52,less than the latter 245.06±13.21. In vitro MRI showed that the T2 signal intensity of HA?PCL@DOX/SPIO and HA?SS?PCL@DOX/SPIO was significantly lower than that of the control group (F values were 613.591 and 569.234,P=0.000), the latter decline rate was more significant. Conclusion The disulfide?bonded hyaluronic acid?functionalized sodium?meter probe for hepatocellular carcinoma has excellent physicochemical properties, good targeting and MRI functions on human hepatoma cell HepG2 at the cellular level in vitro.

Objective To preliminarily explore the anti?cancer efficiency of disulfide?bonded hyaluronic acid?functionalized targeted sodium?meter probe for hepatocellular carcinoma and the feasibility of MRI. Methods Twenty?one nude mice models of subcutaneous liver cancer transplantation were randomly divided into saline, hyaluronic acid?poly ε?caprolactone@ doxorubicin/superparamagnetic iron oxide (HA?PCL@DOX/SPIO) and HA?disulfide?bonded?PCL@DOX/SPIO (HA?SS?PCL@DOX/SPIO) groups, with 7 mice in each group. The experimental groups were injected with micelles at a dose of Fe 5 mg/kg through the tail vein, and the control group was injected with the same amount of saline via the tail vein. MRI was performed before and after injection (2 h, 4 h, 8 h). The T2 value of the region of interest (tumor) was measured and its decline rate was calculated. Twenty?one nude mice models of orthotopic liver cancer transplantation were randomly divided into saline group,HA?PCL@DOX/SPIO and HA?SS?PCL@DOX/SPIO groups, with 7 mice in each group. The experimental groups were injected with micelles at a dose of DOX 2 mg/kg through the tail vein by three consecutive times a day apart, and the control group was injected with the same amount of saline through the tail vein. Continuous observation for 15 days to calculate tumor inhibition rate. One way ANOVA test was used. Results The T2 value of HA?SS?PCL@DOX/SPIO group decreased significantly after 2, 4 and 8 hours (P<0.05) than initial time, which was distinct compared with HA?PCL@DOX/SPIO group. The growth rate of tumor in HA?SS?PCL@DOX/SPIO and HA?PCL@DOX/SPIO groups was significantly lower than that in the control group (F=21.513,P<0.05). The former had the most obvious inhibitory effect on orthotopic liver cancer (47.7% and 28.2%). Conclusion Disulfide?bonded hyaluronic acid?functionalized targeted sodium?meter probe for hepatocellular carcinoma(HA?SS?PCL@DOX/SPIO) has high anti?cancer efficiency and imaging function at the animal level in vivo, and can be used to monitor the early therapeutic effect of tumor at the molecular imaging level.

Objective    To compare the clinical effects of segmentectomy and lobectomy for ≤2 cm lung adenocarcinoma with micropapillary and solid subtype negative by intraoperative frozen sections. Methods    The patients with adenocarcinoma who received segmentectomy or lobectomy in multicenter from June 2020 to March 2021 were included. They were divided into two groups according to a random number table, including a segmentectomy group (n=119, 44 males and 75 females with an average age of 56.6±8.9 years) and a lobectomy group (n=115, 43 males and 72 females with an average of 56.2±9.5 years). The clinical data of the patients were analyzed. Results    There was no significant difference in the baseline data between the two groups (P>0.05). No perioperative death was found. There was no statistical difference in the operation time (111.2±30.0 min vs. 107.3±34.3 min), blood loss (54.2±83.5 mL vs. 40.0±16.4 mL), drainage duration (2.8±0.6 d vs. 2.6±0.6 d), hospital stay time (3.9±2.3 d vs. 3.7±1.1 d) or pathology staging (P>0.05) between the two groups. The postoperative pulmonary function analysis revealed that the mean decreased values of forced vital capacity and forced expiratory volume in one second percent predicted in the segmentectomy group were significantly better than those in the lobectomy group (0.2±0.3 L vs. 0.4±0.3 L, P=0.005; 0.3%±8.1% vs. 2.9%±7.4%, P=0.041). Conclusion    Segmentectomy is effective in protecting lungs function, which is expected to improve life quality of patients.