抗人肝细胞肝癌单克隆抗体的制备及鉴定

Objective To establish and identify a new monoclonal antibody (MAb) against human hepatoceilular carcinoma (HCC) with high specificity and to provide evidence for targeted diagnosis and treatment of liver cancers. Methods Human HCC cell line HepG-2 was used to immunize BALB/c mice using the tail vein injection combined with intrasplenic injection. The sensitized splenic cells were fused with isogenic rat myeloma cells SP2/0-Ag14. The supernatant of the fused cells was detected by ABC immunohistochemistry (IHC) for primary antibody screening. The positive clones were subcloned using the limiting dilution method, and the chromosome of hybridoma was analyzed. The specificity of obtained MAb against HCC was identified by IHC. The epitope of the antigen was detected with laser confocal scanning microscope (LSCM). The subclass of the MAb was analyzed by enzyme linked immunosorbent assay (ELISA). The biodistribution of the MAb was detected in HCC-bearing nude mice to identify its biological characteristics. Results (1) One strain of hybridoma was obtained which produced antihuman HCC MAb with up to 98.5% (67/68) specificity in binding with liver cancer tissues. (2) After injection of 131I-MAb, radioactivity appeared increasingly stronger at the site of tumor and gradually weaker in the blood, liver, kidney and lungs. At 72 h, the ratio of radioactivity in tumor over blood and in tumor over liver was 15.76±3.28 and 7.23±1.70, respectively. Conclusions A novel MAb targeting well at HCC with high specificity is obtained. The MAb may be of potential use in diagnosis and treatment of primary HCC.     

多聚左旋赖氨酸协同超顺磁性氧化铁标记大鼠C6脑胶质瘤细胞

Objective To investigate the labeling rate with poly-L-lysine (PLL) and super paramagnetic iron oxide particles (SPIO) for C6 rat glioma cells and the impact of PLL and SPIO on labeled cell bioactivity. Methods C6 rat glioma cells were incubated without SPIO or PLL (control group), with 25 mg/L SPIO (group A), 25 mg/L SPIO+0.75 mg/L PLL (group B), and 50 mg/L SPIO+1.5 mg/L PLL (group C) , respectively. The labeled cell bioactivity was analyzed by MTS assay (mono-nuclear cell direct cvtotoxicity assay) at different incubation time (6, 24 and 48 h) after treatment, and the labeling rate was detected by Prussian Blue staining. The labeled cells mass were imaged in vitro using a 3.0 T MRI scanner with GRE/30° T2*WI sequence. The R2* values and signal intensity were compared among these groups. Results Up to 48 h post-labeling, there was no significant decrease of cell bioactivity in all labeled cell groups (all P>0.05). Prussian Blue staining showed that the labeling rate was >98% in group B and C compared with about 70% in group A. The staining appeared increasingly darker in SPIO/PLL mixed labeled cells along with higher concentration of SPIO. 3.0 T MRI was signal-sensitive for the labeled cells. The R2* values were 11.76±5.74, 12.13±4.39, 61.22±27.85 and 90.07±35.59 for control group and group A, B and C, respectively. Difference of R2* value was not found between control group and group A (P>0.05), but was found between group B and C, meanwhile R* values of group B and C were significantly different with control group and group A (all P<0.01). Conclusions PLL may enhance the efficiency of SPIO labeling for C6 glioma cells and has no significant impact on the cell bioactivity. 3.0 T MRI with GRE/30° T2*WI sequence is signal-sensitive for labeled cells in vitro. The R2* value may increase along with intracellular SPIO level. 25 mg/L SPIO+0.75 mg/L PLL may yield satisfactory labeling for rat C6 glioma cells in vitro.     

The precursor frequency of alloreactive CTLs is proportional to the number of T cell epitope specificities

The aim of this study is to find the experimental evidence that the precursor frequency of alloreactive CTLs is proportional to the number of the T-cell epitope specificities. The number of T-cell epitope specificities was manipulated by pulsing different number of HLA-A2 restricted peptide(s) onto the T2 cells, which acted as stimulating cells to elicit allo-reaction by co-culturing with peripheral blood lymphocytes (PBLs) of HLA-A2 negative individual. Ten HLA-A2 restricted peptides (all were normal cell components) were synthesized, and cell peptide extract was prepared by frozen and thawed.T2 cells loaded with different number of peptide(s) were co-cultured with PBLs of an HLA-A2 negative individual; the latter were stained with PKH67 in advance. Then the proliferation was monitored with flow cytometry, and the precursor frequency of the effector cells was analyzed by the ModFit Software. After 6 d of culture, no proliferation was observed in the bulk culture of PBL alone, and obvious proliferation took place when PBLs of the HLA-A2 negative were co-cultured with T2 cells loaded with or without loading peptide(s). The precursor frequency of the alloreactive CTLs was 0.052 819 for co-culture with T2 cells loaded without peptide; however it was 0.030 429 for T2 cells with EBV/ LMP2A and 0. 030 528 for T2 cells loaded with a single autogeneic peptide, and increased up to 0.144 942 for T2 cells loaded with 10 autogeneic peptides; the precursor frequency was 0.203 649 when co-cultured with T2 cells loaded with miscellaneous peptides extracted from the cytoplasm of T2 cells. This study reveals that the precursor frequency of alloreactive CTLs is proportional to the number of T-cell epitope specificities, and independent of the density of the allogeneic HLA ClassⅠmolecule. Our findings support the hypothesis that the alloreactive T cell populations comprise miscellaneous T cell clones; each is specific to corresponding pMHC. The novel constellation of peptides presented by allogeneic MHC molecules makes thousands of different epitopes, which account for the exceptional high precursor frequency of alloreactive T cells.     

酵母多糖在Wistar大鼠真菌性眼内炎中的致病作用

Objective To investigate the histopathologic features, changes of blood-ocular barrier and the expression of tumor necrosis factor-α (TNF-β) and interleukin-1β(IL-1β in vitreous body of Wistar rats with zymosan-induced endophthalmitis.Methods Random number table method was used to assign the Wistar rats into saline control group (SC group,n=168) and endophthalmitis group (EO group,n=168).Rats of EO group received intravitreal injection of zymosan to induce endophthalmitis, while the control rats received injection of equivalent-ose sterile saline.The rat eyes were observed for signs of ocular inflammation and enucleated for histopathological examination at 6 h, 12 h, 1d, 2d, 3d, 5d and 7 d after injection respectively.Furthermore, aqueous humor for determination of protein levels and vitreous body for levels of TNF-α and IL-1βwere collected.Results Severe inflammatory response in the eyes was observed in EO group within 6 h to 3 d after zymosan injection, and was basically resolved after day 7.A peak intraocular leucocyte count was observed in EO group on day 1 [(482.63 ±91.15) cells/eye] that rapidly declined on day 3[(131.25±7.95) cells/eye].The increased levels of TNF-α and IL-1βin EO group peaked at 24 h after injection [TNF-α: (331.17±9.81) ng/L, IL-1β (2 156.09± 440.27) ng/L, respectively], persisted to 48 h after injection, and began to decline rapidly afterwards.On day 7 after injection, levels of TNF-α and IL-1βreturned to baseline[TNF-α: (5.55±2.27)ng/L, IL-1∞ (43.66±8.73) ng/L, respectively].Compared with SC group 6 h after injection, significantly higher protein levels in aqueous humor were detected in the EO group at all the time points (all P＜0.01).Conclusions Acute experimental endophthalmitis was successfully induced in the Wistar rats with intravitreal injection of zymosan.Massive leucocyte intraocular infiltration, blood-ocular barrier breakdown and high levels of TNF-α and IL-1βexpression in vitreous body may be the major pathologic characteristics in this experimental endophthalmitis model.     

Three dimensional structure prediction and proton nuclear magnetic resonance analysis of toxic pesticides in human blood plasma

The purpose of this study was to investigate the nuclear magnetic resonance(NMR) assignments of hydrolyzed products extracted from human blood plasma.The correlations between chemical,functional and structural properties of highly toxic pesticides were investigated using the PreADME analysis.We observed that toxic pesticides possessed higher molecular weight and,more hydrogen bond donors and acceptors when compared with less toxic pesticides.The occurrence of functional groups and structural properties was analyzed using 1H-NMR.The 1HNMR spectra of the phosphomethoxy class of pesticides were characterized by methyl resonances at 3.7-3.9 ppm(δ) with the coupling constants of 11-16 Hz(JP-CH3).In phosphoethoxy pesticides,the methyl resonance was about 1.4 ppm(δ) with the coupling constant of 10 Hz(JP-CH2) and the methylene resonances was 4.2-4.4 ppm(δ) with the coupling constant of 0.8 Hz(JP-CH3),respectively.Our study shows that the values of four parameters such as chemical shift,coupling constant,integration and relaxation time correlated with the concentration of toxic pesticides,and can be used to characterise the proton groups in the molecular structures of toxic pesticides.     

Optimization of preparation method of atorvastatin calcium sustained-release microspheres北大核心

BACKGROUND: In recent years, studies have found that statins have significant effects on regulating bone metabolism, repairing bone microstructure, inhibiting inflammation, promoting cell proliferation, repairing vascular endothelium, and regulating signal pathway conduction. OBJECTIVE: To optimize the preparation parameters of atorvastatin calcium sustained-release microspheres, to prepare sustained-release microspheres with large drug loading and regular morphology. METHODS: The bovine serum albumin sustained-release microspheres loaded with atorvastatin calcium were prepared by desolvent method. The main factors affected the preparation of the microspheres were screened out. The four key related factors were bovine serum albumin concentration (40, 70, 100 g/L), pH value (7, 8, 9), dosage of atorvastatin calcium (200, 300, 400 µg), and ethanol addition rate (0.2, 0.5, 1 mL/min). The optimal preparation conditions of large drug loading were screened by orthogonal test. Atorvastatin calcium-loaded bovine serum albumin sustained-release microspheres were prepared under optimal parameters and placed in PBS for sustained-release performance testing. RESULTS AND CONCLUSION: (1) The optimum preparation parameters were as follows. The concentration of bovine serum albumin was 100 g/L; the pH value was 7; the dosage of atorvastatin calcium was 400 µg; the addition rate of ethanol was 0.2 mL/min. (2) The microspheres prepared under this parameter had regular morphology and smooth surface. The particle size was (425.0±13.8) nm and the encapsulation efficiency of drug loaded microspheres was up to 85.70%. The in vitro release time could last for more than 48 hours, and the cumulative release reached 73% which had a relatively good sustained release effect. (3) It is indicated that the stable sustained-release microspheres loaded with atorvastatin calcium were successfully prepared. The sustained-release microspheres with high drug loading and stability can achieve sustained drug release. © 2022, Publishing House of Chinese Journal of Tissue Engineering Research. All rights reserved.     

Evaluation of Coronary Artery Disease Using Technetium-99m-Sestamibi First-Pass and Perfusion Imaging with Dipyridamole Infusion

The aims of this study were: (1) to test whether first-pass radio-nuclide angiocardiography (FPRNA) adds userul information to perfusion scintigraphy; and (2) to assess the relative accuracy of perfusion and functional imaging in combination with dipyndam ole for the evaluation of CAD. Methods: Thirty patients with angiographically proven CAD (17 with prior infaction) were studied on separate days at rest and with dipyridamole infusion (0.7rog/kg over 4 min). Tomographic images were evaluated using an uptake score. Dipyridamole FPRNA was considered positive in case of stress-induced wall motion abnormality or ejection fracticn decrease. Results: The CAD detection rate of perfusion imaging was 100％, while that of FPRNA was 70％ using wall marion criteria, 63％ using ejection fraction response and 77％ condidering any abnormality. 96％ specificity and 82％ accuracy. FPRNA results were 50％, 100％ and 80％ respectively. Perfusion imaging was significantly     

Technetium-99m-labeled annexin V imaging for detecting prosthetic joint infection in a rabbit model

Accurate and timely diagnosis of prosthetic joint infection is essential to initiate early treatment and achieve a favorable outcome.In this study,we used a rabbit model to assess the feasibility of technetium-99m-labeled annexin V for detecting prosthetic joint infection.Right knee arthroplasty was performed on 24 New Zealand rabbits.After surgery,methicillin-susceptible Staphylococcus aureus was intra-articularly injected to create a model of prosthetic joint infection(the infected group,n = 12).Rabbits in the control group were injected with sterile saline(n=12).Seven and 21 days after surgery,technetium-99m-labeled annexin V imaging was performed in 6 rabbits of each group.Images were acquired 1 and 4 hours after injection of technetium-99 mlabeled annexin V(150 MBq).The operated-to-normal-knee activity ratios were calculated for quantitative analysis.Seven days after surgery,increased technetium-99m-labeled annexin V uptake was observed in all cases.However,at 21 days a notable decrease was found in the control group,but not in the infected group.The operated-to-normal-knee activity ratios of the infected group were 1.84 ± 0.29 in the early phase and 2.19 ±0.34 in the delay phase,both of which were significantly higher than those of the control group(P=0.03 and P=0.02).The receiver operator characteristic curve analysis showed that the operated-to-normal-knee activity ratios of the delay phase at 21 days was the best indicator,with an accuracy of 80%.In conclusion,technetium-99m-labeled annexin V imaging could effectively distinguish an infected prosthetic joint from an uninfected prosthetic joint in a rabbit model.     

体内电穿孔在质粒介导的基因转移及DNA免疫中的应用

Objective To investigate the effects of in vivo electroporation on plasmid mediated reporter gene expression and immunogenicity of DNA vaccine. Methods Luciferase expression plasmid was administered intramuscularly to BALB/c mice at 8μg and 40μg dosage level through injection with or without eletroporation Luciferase expression level in murine muscle was detected by IVIS imaging system 24 h after injection. DNA vaccine plasmid p1.0-gp1455m carrying codon-optimized env gene of CN54 strain ( HIV-1 CRF07_BC) was administered to mice at dosages of 8μg and 40μg through the two approaches mentioned above. Mice were immunized at week 0,2 and 4. Env-specific immune responses were detected at two weeks post the second and the third vaccinations. Env-specific antibody immune responses were determined by ELISA. Euv-specific cellular immune responses were determined by IFN-γ ELISPOT. Results Luciferase expression level in murine muscle was significantly increased as much as 35 folds through in vivo eletroporation. Results of ELISA and ELISPOT revealed that in vivo eletroporation could significantly enhance both the humoral and cellular immune responses induced by DNA vaccination. The responses induced by electrodelivered p1.0-gp1455m at 8 μg dosage were better than those induced by simple intramuscular injection with 40 μg of plasmid DNA. On the other hand, 2 injections followed by electroporation elicited comparable level of humoral and cellular immune responses with those induced by 3 injections without electroporation. Conclusion In vivo electroporation was capable of enhancing both the plasmid-mediated gene expression and immunogenicity of DNA vaccine.     

体内电穿孔在质粒介导的基因转移及DNA免疫中的应用

Objective To investigate the effects of in vivo electroporation on plasmid mediated reporter gene expression and immunogenicity of DNA vaccine. Methods Luciferase expression plasmid was administered intramuscularly to BALB/c mice at 8μg and 40μg dosage level through injection with or without eletroporation Luciferase expression level in murine muscle was detected by IVIS imaging system 24 h after injection. DNA vaccine plasmid p1.0-gp1455m carrying codon-optimized env gene of CN54 strain ( HIV-1 CRF07_BC) was administered to mice at dosages of 8μg and 40μg through the two approaches mentioned above. Mice were immunized at week 0,2 and 4. Env-specific immune responses were detected at two weeks post the second and the third vaccinations. Env-specific antibody immune responses were determined by ELISA. Euv-specific cellular immune responses were determined by IFN-γ ELISPOT. Results Luciferase expression level in murine muscle was significantly increased as much as 35 folds through in vivo eletroporation. Results of ELISA and ELISPOT revealed that in vivo eletroporation could significantly enhance both the humoral and cellular immune responses induced by DNA vaccination. The responses induced by electrodelivered p1.0-gp1455m at 8 μg dosage were better than those induced by simple intramuscular injection with 40 μg of plasmid DNA. On the other hand, 2 injections followed by electroporation elicited comparable level of humoral and cellular immune responses with those induced by 3 injections without electroporation. Conclusion In vivo electroporation was capable of enhancing both the plasmid-mediated gene expression and immunogenicity of DNA vaccine.     

Pain and Effusion and Quadriceps Activation and Strength

Context:

Quadriceps dysfunction is a common consequence of knee joint injury and disease, yet its causes remain elusive.

Objective:

To determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion affect the magnitude of quadriceps dysfunction.

Design:

Crossover study.

Setting:

University research laboratory.

Patients or Other Participants:

Fourteen (8 men, 6 women; age = 23.6 ± 4.8 years, height = 170.3 ± 9.16 cm, mass = 72.9 ± 11.84 kg) healthy volunteers.

Intervention(s):

All participants were tested under 4 randomized conditions: normal knee, effused knee, painful knee, and effused and painful knee.

Main Outcome Measure(s):

Quadriceps strength (Nm/kg) and activation (central activation ratio) were assessed after each condition was induced.

Results:

Quadriceps strength and activation were highest under the normal knee condition and differed from the 3 experimental knee conditions (P < .05). No differences were noted among the 3 experimental knee conditions for either variable (P > .05).

Conclusions:

Both pain and effusion led to quadriceps dysfunction, but the interaction of the 2 stimuli did not increase the magnitude of the strength or activation deficits. Therefore, pain and effusion can be considered equally potent in eliciting quadriceps inhibition. Given that pain and effusion accompany numerous knee conditions, the prevalence of quadriceps dysfunction is likely high.Key Words: arthrogenic muscle inhibition, central activation failure, voluntary activation, muscles

Key Points

• Knee pain and effusion resulted in arthrogenic muscle inhibition and weakness of the quadriceps.
• The simultaneous presence of pain and effusion did not increase the magnitude of quadriceps dysfunction.
• To reduce arthrogenic muscle inhibition and improve muscle strength, clinicians should employ interventions that target removing both pain and effusion.

Quadriceps weakness is a common consequence of traumatic knee joint injury^1,2 and chronic degenerative knee joint conditions.^3,4 Arthrogenic muscle inhibition (AMI), a neurologic decline in muscle activation, results in quadriceps weakness and hinders rehabilitation by preventing gains in strength.⁵ The inability to reverse AMI and restore muscle function can lead to decreased physical abilities,⁶ biomechanical deficits,⁷ and possibly reinjury.⁵ Furthermore, researchers^8,9 have suggested that quadriceps weakness resulting from AMI may place patients at risk for developing osteoarthritis in the knee. In light of the substantial influence of quadriceps AMI on these clinically relevant outcomes, we need to improve our understanding of the factors that contribute to this neurologic decline in muscle activity so efforts to target and reverse it can be implemented and gains in strength can be achieved more easily.Joint injury and disease are accompanied by numerous sequelae (ie, pain, swelling, tissue damage, inflammation), so ascertaining which one ultimately leads to neurologic muscle dysfunction is difficult. Whereas a joint effusion can result in AMI,^10–12 the effects of pain are less understood despite many clinicians attributing AMI to pain. Using techniques that introduce knee pain without accompanying injury may provide insights into the role of pain in eliciting AMI.The degree of knee joint damage may play a role in the quantity of AMI that manifests. Hurley et al^13,14 demonstrated that quadriceps AMI, measured using an interpolated-twitch technique, was greater in patients with extensive traumatic knee injury (eg, fractured tibial plateau, ruptured medial collateral ligament, and medial meniscectomy) than patients with isolated joint trauma (ie, isolated anterior cruciate ligament [ACL] rupture). Similarly, patients with more knee joint symptoms (ie, greater number of symptoms and increased severity of symptoms) may present with greater magnitudes of quadriceps inhibition. Recently, investigators¹⁵ have suggested that patients with more pain display less quadriceps strength, supporting this tenet. Given that effusion and pain often present simultaneously with joint injuries and diseases, such as ACL injury and osteoarthritis, examining both the isolated and cumulative effects of these sequelae appears warranted to determine if they influence the magnitude of muscle inhibition.Experimental joint-effusion and pain models are safe and effective experimental methods that allow for the isolated examination of their effects on muscle function. The effusion model, whereby sterile saline is injected directly into the knee joint capsule,⁷ produces a clinically relevant magnitude of the joint effusion that may be present with traumatic injury. Effusion is thought to activate group II afferents responding to stretch or pressure,^16–18 which in turn may facilitate group Ib interneurons and result in quadriceps AMI.⁵ The pain model involves injecting hypertonic saline into the infrapatellar fat pad to produce anteromedial knee pain similar to that described in patients with patellofemoral pain syndrome.¹⁹ Pain is considered to initiate AMI through activation of group III and IV afferents that act as nocioceptors to signal damage or potential damage to joint structures.^16–18 The firing of these afferents then may lead to facilitation of group Ib interneurons, the flexion reflex, or the gamma loop, ultimately resulting in quadriceps inhibition.²⁰ Thus, these models allow us to create symptoms that are associated with knee injury and have the added benefit of providing a way to examine their effects in isolation.Therefore, the purpose of our study was to determine the effects of pain on quadriceps strength and activation and to learn if simultaneous pain and knee joint effusion would affect the magnitude of quadriceps dysfunction. We hypothesized that pain alone would result in quadriceps inhibition and that the magnitude of inhibition would be greater when effusion and pain were present simultaneously.     

即早基因c-fos与脑血管病及学习记忆

即早基因c-fos是广泛存在于原核细胞和真核细胞的高度保守基因.在正常情况下,c-fos基因参与细胞生长、分化、信息传递、学习和记忆等生理过程,而在病理情况下c-fos基因表达及调控变化与多种疾病的发生和发展有关.C-fos在中枢神经系统的某些部位可有基础水平的表达,但表达很低,当受到如脑缺血、脑出血、痫性发作、应激等刺激后,其在数十分钟内做出反应,在对外界刺激-转录耦联的信忠传递过程中起着核内第三信使的重要作用.     

Opportunities and challenges in the prevention and control of cancer and other chronic diseases: children's diet and nutrition and weight and physical activity

OBJECTIVE: The purpose of this article is to review the role of behavioral research in disease prevention and control, with a particular emphasis on lifestyle- and behavior-related cancer and chronic disease risk factors--specifically, relationships among diet and nutrition and weight and physical activity with adult cancer, and tracking developmental origins of these health-promoting and health-compromising behaviors from childhood into adulthood. METHOD: After reviewing the background of the field of cancer prevention and control and establishing plausibility for the role of child health behavior in adult cancer risk, studies selected from the pediatric published literature are reviewed. Articles were retrieved, selected, and summarized to illustrate that results from separate but related fields of study are combinable to yield insights into the prevention and control of cancer and other chronic diseases in adulthood through the conduct of nonintervention and intervention research with children in clinical, public health, and other contexts. RESULTS: As illustrated by the evidence presented in this review, there are numerous reasons (biological, psychological, and social), opportunities (school and community, health care, and family settings), and approaches (nonintervention and intervention) to understand and impact behavior change in children's diet and nutrition and weight and physical activity. CONCLUSIONS: Further development and evaluation of behavioral science intervention protocols conducted with children are necessary to understand the efficacy of these approaches and their public health impact on proximal and distal cancer, cancer-related, and chronic disease outcomes before diffusion. It is clear that more attention should be paid to early life and early developmental phases in cancer prevention.