Objective: To construct a phage display library of human single-chain Fv antibodies against blood group Rh(D) substance. Methods: Combining phage display library techniques, isolated total RNA from B lymphoblastoid cell lines secreting anti-Rh(D) antibodies was used for the synthesis of the first strand of cDNA, V_ H and V_ L genes were amplified by 2nd PCR and linked together by splicing overlap extension (SOE) with the use of a (Gly_ 4Ser)_ 3 linker. The resulted scFv genes were then cloned into pCANTAB5E vectors and displayed on the phage. Phage clones were selected using intact red cells as a source of antigen. After 4 rounds of "binding-elution-enrichment", each clone was assayed for specificity by Dot ELISA. Results: A phage antibody library, with the sink size being 1.2?107, was obtained. The percentage of full-length scFv gene inserted into phage DNA was 0.80. Rescued by helper phage, a phage scFv library with titer of 3?108 pfu/ml was established. Specific phages with scFv were acquired after 4 rounds of panning, one clone exhibiting specific binding to Rh+ cell was identified by Dot ELISA. Conclusion: A strategy for construction phage antibody library by means of phage display technique was practicable, which would be useful in screening engineered antibodies against human Rh (D) blood group substances.

Objective: To develop Epstein-Barr virus(EBV)-transformed human peripheral blood B cell lines from healthy volunteers of type B.Methods: B lymphocytes from healthy volunteers of type B capable of producing anti-A antibodies were transformed by EBV,while Cyclosporine A(CsA),as an immunosuppressive agent that could selectively inhibit T-lymphocytes and protect B-lymphocytes from regression,was used in the experiment.Then,the supernanant of the cell culture medium was tested with red blood cells of type A,B and O by agglutinin assay.Results: Twelve of the15 EBV-transformed B lymphocyte cell lines were established,with a success rate of 80%,while 9 human B lymphocyte lines secreted anti-A antibodies.Conclusion: Human B lymphocyte lines secreting antibodies to A antigens were successfully developed,which helps further studies of human blood specific monoclonal antibodies.

Objective To explore the application value of pulmonary artery monitoring program for intracranial and cervical artery angiography with dual-energy CT.Methods Sixty patients performed intracranial and cervical artery angiography with dual-energy CT were divided into two groups according to the random number table.Group A (optimization group,30 patients):the monitoring points were located in the main pulmonary artery,with threshold 150 HU,trigger delay time 8-9 s,pitch 0.9,and the iohexol (350 mg I/ml) 60-65 ml.Group B (conventional group,30 patients):the monitoring points were located in aortic arch,with threshold 100 HU,trigger delay time 5 s,and the iohexol (350 mg I/ml)60-70 ml.Patients with the body weight less than 75 kg were injected with the flow rate of 4.0 ml/s,and those weight greater than 75 kg or with body mass index (BMI) greater than 27 kg/m2 were 4.5 ml/s,following 40 ml saline solution with the same flow rate respectively.All images were transferred to Siemens Syngo workstation for further processing and analysis.The attenuation values were measured on axial images in the common carotid artery,internal and external carotid artery,vertebral and basilar artery,the horizontal segment of the middle cerebral artery,the subclavian vein of injection side,the proximal,middle and distal segment of jugular vein,the straight sinus and the superior sagittal sinus.Two experienced observers blinded independently evaluated the image quality of CTA,the impact of contrast material residues artifacts of subclavian vein of injected side and the affect of venous return factors on arterial image display.Images of volume rendering technique(VR),the maximum intensity projection (MIP),and curved planar reformation (CPR) were reconstructed using dual energy bone removal and Inspace and 3D software.The mean intraluminal attenuation of contrast material (HU),the volume of contrast material and the mean image quality scores were compared with t test between the two groups.Chi-square test was used to compare of image contrast agents residual artifacts,the absence segments of the root of the neck artery,and the degree of the jugular vein return in two groups.Results The mean CT values were 372-414 HU for each segment of artery showing no significant differences between two groups (P ＞ 0.05).In group A,the CT values of the jugular vein in proximal,middle,distal segment of the two sides were (95 ± 36),(95 ± 36),(131 ±58),(133 ± 57),(174 ± 68),and (180 ± 66) HU respectively.In group B,CT values were (135 ± 58),(137 ±59),(170 ±58),(181 ±58),(218 ±62),and (224 ±68) HU.The CT value of jugular venous in group A was significantly lower than that in group B (t =-3.30--2.54,P ＜ 0.05).Case numbers of contrast agent residual artifacts of injection side subclavian vein and the absence segments of root of the neck artery at the beginning in group A (5,11) were less than that in group B (12,24) (x2 =4.02,5.65,P ＜0.05).The degree of the jugular vein return in group A were lesser severely than that in group B (x2 =6.79,6.37,P ＜0.05).Below the level of carotid artery bifurcation:slight 5 patients,severe 1 patient in group A vs.slight 15 patients,severe 9 patients in group B.Above the level of carotid artery bifurcation:slight 9 patients,severe 6 patients in group A vs.slight 12 patients,severe 17 patients in group B.Image quality scores of group A (3.84 ± 0.40) was higher than that of group B (3.64 ± 0.63) (t =4.26,P ＜0.05).Conclusions Pulmonary Artery Monitoring combines with 60-65 ml contrast material optimization scheme for intracranial and cervical artery angiography with dual energy CT can significantly reduce the degree of jugular venous return and contrast material residues artifacts of subclavian vein of injected side.This technique is helpful to improve the work efficiency and image quality.

Objective To explore the value of the ultrasound contrast agent in conjunction with advanced contrast imaging techniques in the evaluation of the renal cortical perfusion. Methods The animal model was established with ligating renal artery to cause the renal cortical perfusion decrease of rabbits. Real-time harmonic gray scale imaging was performed to visualize the contrast enhancement of the renal cortex. After bolus injection of contrast agent SonoVue, dynamic image was observed and recorded during the first three minutes. According to the time-intensity curve (TIC), accelerating time (AT),Peak intensity (A), time to half of peak intensity (T), difference between peak and base (PBD), slope rate of the curve (β) and the product of A and β (A·β) were obtained before and after the renal artery operation. The t test and correlation analysis was used to examine the above parameters. Results Before the renal artery operation, renal cortex enhanced rapidly and obviously, the way of the enhancement was renal artery-cortex-pyramids. After ligation of renal artery, AT prolonged evidently (P<0.001), A decreased obviously (P<0.001), T obviously shortened (P<0.001), PBD decreased markedly (P<0.001), while the decline of βinduced (P<0.002) and A·βobvious dropped(P<0.001). AT, BPD and A·βcorrelated with stenosis level of renal artery. Compared with 31%－69% group, AT prolonged evidently (P<0.001). A decreased obviously (P<0.029), PBD decreased obviously (P<0.001), A·β was dropped markedly (P<0.02) in 70%-93% stenosis group. Conclusions Contrast agent combining with developed contrast specific imaging is able to detect changes of renal cortical microcirculation sensitively with the help of analysis of TIC.

OBJECTIVETo study the effect of Salvia miltiorrhiza on alveolar bone metabolism and variation in bone mass in diabetic rats, in order to detect whether it has an inhibitory effect on alveolar bone osteoporosis caused by diabetics.

METHODIntraperitoneal injection of alloxan induced diabetes in rats. After one week of observation and maintenance of stable blood sugar level, they were treated with S. miltiorrhiza. The rats were sacrificed at the eighth week after fasting for 12 h and blood samples were collected for analysis of blood glucose and rate of bone metabolism. Meanwhile, their alveolar bones were collected for determining bone mineral density (BMD) and histological sections were made for histomorphology observation.

RESULTDiabetic rats showed varying degrees of abnormality in bone metabolism indicators and significant reduction in bone mineral density. After treatment with S. miltiorrhiza, their symptoms reduced to some extend and all indicators were improved especially bone density.

CONCLUSIONS. miltiorrhiza has a certain inhibitory effect on alveolar bone osteoporosis in diabetic rats in early stage.

Animals ; Bone Density ; drug effects ; Diabetes Mellitus ; metabolism ; pathology ; physiopathology ; Drugs, Chinese Herbal ; pharmacology ; Male ; Maxilla ; drug effects ; metabolism ; pathology ; Organ Size ; drug effects ; Rats ; Rats, Wistar ; Salvia miltiorrhiza ; chemistry

Recently,optical biometers based on swept-source optical coherence tomography (SS-OCT) technology have been applied to clinical ophthalmology.Compared with previous optical biometers,SS-OCT instruments have many advantages,such as faster processes,more accurate results,higher detection rate,and more built-in formulas for calculating the intraocular lens (IOL) power;moreover,SS-OCT images can be used to detect preliminary maculopathy and predict postoperative tilt of IOL.In this paper,we reviewed the application of SS-OCT biometer in cataract,mainly focus on the principle of work,the evaluation of repeatability and reproducibility,the consistency comparison with other biometers,and the application of SS-OCT in other aspects of cataract.

Objective:To investigate the expression of connexin-43 (Cx43) in steroid-induced osteonecrosis of femoral head and osteoblasts in rats and its regulation mechanism.Methods:The model of steroid-induced osteonecrosis of femoral head (SIONFH) of rat was established. Micro-CT and HE staining were used to observe the degree of bone trabecular destruction and the incidence of empty lacunae. The expression levels of Cx43 and PI3K/Akt signaling pathway related molecules and osteoblast-related proteins in model group and control group were detected by RT-PCT and Western blot. The osteoblast (OB) of rats was further isolated and cultured in vitro. Under treatment of dexamethasone (Dex), Cx43 expression in OB cells was detected by Western blot and immunofluorescence. Western blot was used to detect the effect of glucocorticoid (GC) on the expression of related molecules of PI3K/Akt/β-catenin signaling pathway. Akt activator (SC79) and PI3K inhibitor (LY294002) were used to study the molecular mechanism of Dex regulation on Cx43 expression in OB cells. The regulatory relationship between β-catenin and Cx43 was investigated by immunoprecipitation and small interfere RNA (siRNA) technology.Results:The model of SIONFH in rats was successfully established, which proved that Cx43 expression level in the SIONFH model group was significantly lower than that in the control group, and the expression level of Cx43 was positively correlated with the expression of PI3K/Akt signaling pathway related molecules and osteoblast-related proteins Runx2, ALP and Collagen I Type (COL). In addition, in vitro culture of isolated rat OB cells, the expression of Cx43, p-PI3K, P-Akt and β-catenin in OB cells decreased gradually as the Dex action time went on. Moreover, SC79 pretreatment could significantly reverse the inhibitory effect of GCs on Cx43 expression, while LY294002 could significantly enhance the inhibitory effect of GCs on Cx43. In addition, the immunoprecipitation results showed that β-catenin expression was closely related to Cx43 expression, and further studies showed that β-catenin-siRNA could significantly down-regulate the expression of Cx43.Conclusion:Under the action of GC, the expression level of Cx43 in bone tissue and OB cells decreased significantly, and the possible mechanism was that GCs inhibited the expression of Cx43 by inhibiting the PI3K/Akt/β-catenin signaling pathway, which laid a new theoretical foundation for the further study of the role of Cx43 in the pathogenesis of steroid-induced femoral head necrosis.

Objective: To evaluate the repeatability and reproducibility of swept-source optical coherence tomography biometrics (Tomey OA-2000) for measuring the biological parameters of the eyeballs in healthy people. Methods: A diagnostic test was carried out in this study.One hundred and three healthy volunteers who were recuited from July to September 2018 in Affiliated Hospital of North Sichuan Medical College were included, and the right eyes were examined.Two examiners used OA-2000 for 3 consecutive measurements, respectively.One week later, the first examiner used OA-2000 for 3 consecutive measurements.The ocular biological parameters including axial length (AL), anterior chamber depth (ACD), corneal curvature (K), lens thickness (LT), central corneal thickness (CCT), pupil diameter (PD), and white to white (WTW) were measured.The repeatability and reproducibility of each parameter based on within-subject standard deviation (S_W), repeatability limit of standard deviation (2.77S_W), coefficient of variation (CoV) and intra-class correlation coefficient (ICC) was evaluated.This study followed the Declaration of Helsinki.This study protocol was approved by Ethic Committee of the Affiliated Hospital of North Sichuan Medical College (No.2018ER[A]019). Results: Of the ocular biological parameters measured by OA-2000, CoV was less than 2% while ICC was greater than 0.950, except PD and WTW.The repeatability and reproducibility of K, AL and ACD were all good.For K₁(ø=2.5 mm and 3.0 mm), the S_W was less than 0.120 D, the 2.77S_W was less than 0.350 D, the CoV was less than 0.293%, and the ICC was greater than 0.990; For K₂ (ø = 2.5 mm and 3.0 mm) , the S_W were less than 0.175 D, the 2.77S_W were less than 0.485 D, the CoV was less than 0.396%, the ICC was greater than 0.980; for K_m (ø = 2.5 mm and 3.0 mm), the S_W were less than 0.099 D, 2.77S_W were less than 0.275 D, CoV was less than 0.227%, ICC was greater than 0.996; ACD S_W was less than 0.024 mm, 2.77S_W was less than 0.066 mm, CoV was less than 0.668%, ICC were greater than 0.993; the S_W of AL was less than 0.014 mm, the 2.77S_W was less than 0.038 mm, the CoV was less than 0.057%, and all of the ICCs were 1.000. Conclusions Except PD and WTW, the ocular biological parameters measured by OA-2000 have good repeatability and reproducibility, in which the accuracy of AL measurement is the highest.

Effective bone repair and regeneration is crucial for treating skeletal tissue defects, including osteonecrosis, nonunion fractures, osteoporosis, and various other bone deficiencies. Exosomes, as cellular secretory vesicles, are pivotal in mediating intercellular communication through their cargo of proteins, lipids, and nucleic acids. Mesenchymal stem cell-derived exosomes, in particular, have emerged as promising agents in bone repair and regeneration, showing potential for practical application and clinical translation. Nonetheless, their functional capacity and therapeutic efficacy require enhancement. This review delineates exosome optimization strategies aimed at augmenting secretion and functionality, alongside the incorporation of exosome-functionalized biomaterials for bone healing. Evidence indicates that physical stimulation, molecular interventions, and small-molecule or biomaterial stimuli are effective in increasing exosome output. Moreover, engineering exosomes and their parental cells can further potentiate their therapeutic function. The amalgamation of exosomes with biomaterials represents a burgeoning approach in bone tissue engineering, offering novel therapeutic avenues. This comprehensive analysis aims to guide future applications and the clinical adoption of exosomes in bone tissue restoration.

Objective:To investigate the effects of Connexin-43 (Cx43) on osteoblasts proliferation and osteogenic differentiation and its regulatory mechanism.Methods:Osteoblasts were isolated and cultured in vitro. The osteogenic activity of osteoblasts was detected by alizarin red staining and alkaline phosphatase (ALP) staining after dexamethasone treatment. The expression of Cx43, Runt-related transcription factor 2 (Runx2), ALP, collagen I type (COL-I) and proliferation-related proteins PCNA and CDK4 in osteoblasts were detected by Western-blot. The expressions of osteoblast proteins were detected by immunofluorescence staining. The proliferation of osteoblasts was detected by CCK8 assay. The lentivirus-mediated Cx43 gene overexpression plasmid (Lv-Cx43) was constructed and transfected into osteoblasts. The osteogenic activity and proliferation ability of osteoblasts were further detected by the above methods. Cx43 in osteoblasts was overexpressed by pretreating PD98059. The osteogenic activity and proliferation of Cx43 in overexpressed osteoblasts was detected by CCK8 and alizarin red staining.Results:The isolated osteoblasts have osteogenic differentiation ability. Compared with the control group, 1×10 -6 mol/L dexamethasone treatment could reduce the formation of calcium nodules in osteoblasts. With the increase of dexamethasone treatment duration, the protein expression of Cx43, Runx2, ALP and COL-I in osteoblasts decreased gradually, while the expression of PCNA, CDK4 and p-ERK1/2 decreased. The OD values of normal osteoblasts at 0, 1, 2, 3 and 4 d were 0.316±0.043, 0.891±0.623, 1.683±0.154, 2.315±0.721 and 2.891±0.323, respectively. However, The OD values of osteoblasts treated with dexamethasone were 0.376±0.021, 0.657±0.121, 1.124±0.285, 1.521±0.272, 1.987±0.584, respectively. OD values of dexamethasone treated osteoblasts were lower than those of normal group at 2, 3 and 4 days ( P<0.05). The relative expression levels of Cx43 mRNA in control group, Lv-NC group and Lv-Cx43 group were 0.541±0.086, 0.598±0.018 and 1.000±0.082, respectively. The mRNA expression level of Cx43 in Lv-Cx43 group was higher than that in control group and Lv-NC group ( P<0.05). The ratio of Cx43 protein band to the gray value of GAPDH band in control group, Lv-NC group and Lv-Cx43 group were 0.816±0.737, 0.738±0.643 and 1.145±1.101, respectively. The expression level of Lv-Cx43 was higher than that in control group and Lv-NC group ( P<0.05). The expressions of Runx2, ALP, COL-I mRNA and related marker proteins in Lv-Cx43 group were higher than those in control group and Lv-NC group ( P<0.05). The number of calcium nodules in the Lv-Cx43 group was significantly higher than that in the control group and Lv-NC group. The OD value of osteoblasts and the number of calcium nodules in Lv-Cx43+PD98059 group were significantly lower than those in Lv-Cx43 group ( P<0.05). Conclusion:The proliferation and differentiation ability of osteoblasts is significantly decreased after the treatment of dexamethasone with decreased expression of Cx43. Overexpression of Cx43 can promote the proliferation and osteogenic differentiation of osteoblasts, which may be regulated through the ERK1/2 pathway.