Exosomal miR-23b from bone marrow mesenchymal stem cells alleviates oxidative stress and pyroptosis after intracerebral hemorrhage

Our previous studies showed that miR-23b was downregulated in patients with intracerebral hemorrhage(ICH). This indicates that miR-23b may be closely related to the patho-physiological mechanism of ICH, but this hypothesis lacks direct evidence. In this study, we established rat models of ICH by injecting collagenase Ⅶ into the right basal ganglia and treating them with an injection of bone marrow mesenchymal stem cell(BMSC)-derived exosomal miR-23b via the tail vein. We found that edema in the rat brain was markedly reduced and rat behaviors were improved after BMSC exosomal miR-23b injection compared with those in the ICH groups. Additionally, exosomal miR-23b was transported to the microglia/macrophages, thereby reducing oxidative stress and pyroptosis after ICH. We also used hemin to mimic ICH conditions in vitro. We found that phosphatase and tensin homolog deleted on chromosome 10(PTEN) was the downstream target gene of miR-23b, and exosomal miR-23b exhibited antioxidant effects by regulating the PTEN/Nrf2 pathway. Moreover, miR-23b reduced PTEN binding to NOD-like receptor family pyrin domain containing 3(NLRP3) and NLRP3 inflammasome activation, thereby decreasing the NLRP3-dependent pyroptosis level. These findings suggest that BMSC-derived exosomal miR-23b exhibits antioxidant effects through inhibiting PTEN and alleviating NLRP3 inflammasome-mediated pyroptosis, thereby promoting neurologic function recovery in rats with ICH.

Migration of bone marrow progenitor cells in the adult brain of rats and rabbits

Neurogenesis takes place in the adult mammalian brain in three areas:Subgranular zone of the dentate gyrus(DG);subventricular zone of the lateral ventricle;olfactory bulb.Different molecular markers can be used to characterizethe cells involved in adult neurogenesis.It has been recently suggested that a population of bone marrow(BM)progenitor cells may migrate to the brain and differentiate into neuronal lineage.To explore this hypothesis,we injected recombinant SV40-derived vectors into the BM and followed the potential migration of the transduced cells.Long-term BM-directed gene transfer using recombinant SV40-derived vectors leads to expression of the genes delivered to the BM firstly in circulating cells,then after several months in mature neurons and microglial cells,and thus without central nervous system(CNS)lesion.Most of transgene-expressing cells expressed NeuN,a marker of mature neurons.Thus,BM-derived cells may function as progenitors of CNS cells in adult animals.The mechanism by which the cells from the BM come to be neurons remains to be determined.Although the observed gradual increase in transgene-expressing neurons over 16mo suggests that the pathway involved differentiation of BM-resident cells into neurons,cell fusion as the principal route cannot be totally ruled out.Additional studies using similar viral vectors showed that BM-derived progenitor cells migrating in the CNS express markers of neuronal precursors or immature neurons.Transgene-positive cells were found in the subgranular zone of the DG of the hippocampus 16 mo after intramarrow injection of the vector.In addition to cells expressing markers of mature neurons,transgene-positive cells were also positive for nestin and doublecortin,molecules expressed by developing neuronal cells.These cells were actively proliferating,as shown by short term BrdU incorporation studies.Inducing seizures by using kainic acid increased the number of BM progenitor cells transduced by SV40vectors migrating to the hippocampus,and these cells were seen at earlier time points in the DG.We show that the cell membrane chemokine receptor,CCR5,and its ligands,enhance CNS inflammation and seizure activity in a model of neuronal excitotoxicity.SV40-based gene delivery of RNAi targeting CCR5 to the BM results in downregulating CCR5 in circulating cells,suggesting that CCR5 plays an important role in regulating traffic of BM-derived cells into the CNS,both in the basal state and in response to injury.Furthermore,reduction in CCR5 expression incirculating cells provides profound neuroprotection from excitotoxic neuronal injury,reduces neuroinflammation,and increases neuronal regeneration following this type of insult.These results suggest that BM-derived,transgeneexpressing,cells can migrate to the brain and that they become neurons,at least in part,by differentiating into neuron precursors and subsequently developing into mature neurons.

miR-23a/b regulates the balance between osteoblast and adipocyte differentiation in bone marrow mesenchymal stem cells

Age-related osteoporosis is associated with the reduced capacity of bone marrow mesenchymal stem cells （BMSCs） to differentiate into osteoblasts instead of adipocytes. However, the molecular mechanisms that decide the fate of BMSCs remain unclear. In our study, microRNA-23a, and microRNA-23b （miR-23a/b） were found to be markedly downregulated in BMSCs of aged mice and humans. The overexpression of miR-23a/b in BMSCs promoted osteogenic differentiation, whereas the inhibition of miR-23a/b increased adipogenic differentiation. Transmembrane protein 64 （Tmem64）, which has expression levels inversely related to those of miR-23a/b in aged and young mice, was identified as a major target of miR-23a/b during BMSC differentiation. In conclusion, our study suggests that miR-23a/b has a critical role in the regulation of mesenchymal lineage differentiation through the suppression of Tmem64.

Factors affecting directional migration of bone marrow mesenchymal stem cells to the injured spinal cord

Microtubule-associated protein 1B plays an important role in axon guidance and neuronal migration. In the present study, we sought to discover the mechanisms underlying microtu- bule-associated protein 1B mediation of axon guidance and neuronal migration. We exposed bone marrow mesenchymal stem cells to okadaic acid or N-acetyl-D-erythro-sphingosine （an inhibitor and stimulator, respectively, of protein phosphatase 2A） for 24 hours. The expression of the phosphorylated form of type I microtubule-associated protein 1B in the cells was greater after exposure to okadaic acid and lower after N-acetyl-D-erythro-sphingosine. We then injected the bone marrow mesenchymal stem cells through the ear vein into rabbit models of spinal cord contusion. The migration of bone marrow mesenchymal stem cells towards the injured spinal cord was poorer in cells exposed to okadaic acid- and N-acetyl-D-erythro-sphingosine than in non-treated bone marrow mesenchymal stem cells. Finally, we blocked phosphatidylinosi- tol 3-kinase （PI3K） and extracellular signal-regulated kinase 1/2 （ERK1/2） pathways in rabbit bone marrow mesenchymal stem cells using the inhibitors LY294002 and U0126, respectively. LY294002 resulted in an elevated expression of phosphorylated type I microtubule-associated protein 1B, whereas U0126 caused a reduction in expression. The present data indicate that PI3K and ERKI/2 in bone marrow mesenchymal stem cells modulate the phosphorylation of micro- tubule-associated protein 1B via a cross-signaling network, and affect the migratory efficiency of bone marrow mesenchymal stem cells towards injured spinal cord.

Outcomes of autologous bone marrow mononuclear cell transplantation in decompensated liver cirrhosis

AIM: To determine the long-term efficacy of autologous bone marrow mononuclear cells (BM-MNCs) transplantation in terms of improving liver function and reducing complications in patients with decompensated cirrhosis.

Exosomes derived from bone marrow mesenchymal stem cells inhibit neuroinflammation after traumatic brain injury

Exosomes derived from bone marrow mesenchymal stem cells can inhibit neuroinflammation through regulating microglial phenotypes and promoting nerve injury repair.However,the underlying molecular mechanism remains unclear.In this study,we investigated the mechanism by which exosomes derived from bone marrow mesenchymal stem cells inhibit neuroinflammation.Our in vitro co-culture experiments showed that bone marrow mesenchymal stem cells and their exosomes promoted the polarization of activated BV2 microglia to their anti-inflammatory phenotype,inhibited the expression of proinflammatory cytokines,and increased the expression of anti-inflammatory cytokines.Our in vivo experiments showed that tail vein injection of exosomes reduced cell apoptosis in cortical tissue of mouse models of traumatic brain injury,inhibited neuroinflammation,and promoted the transformation of microglia to the anti-inflammatory phenotype.We screened some microRNAs related to neuroinflammation using microRNA sequencing and found that microRNA-181b seemed to be actively involved in the process.Finally,we regulated the expression of miR181b in the brain tissue of mouse models of traumatic brain injury using lentiviral transfection.We found that miR181b overexpression effectively reduced apoptosis and neuroinflamatory response after traumatic brain injury and promoted the transformation of microglia to the anti-inflammatory phenotype.The interleukin 10/STAT3 pathway was activated during this process.These findings suggest that the inhibitory effects of exosomes derived from bone marrow mesenchymal stem cells on neuroinflamation after traumatic brain injury may be realized by the action of miR181b on the interleukin 10/STAT3 pathway.

Differentiation of Bone Marrow Mesenchymal Stem Cells into Retinal Ganglion-like Cells Induced by Math5

We explored the appropriate inducing conditions needed to facilitate the differentiation of bone marrow mesenchymal stem cells（BMSCs） into retinal ganglion cells（RGCs）.Math5,a pro-neural basic helixloop-helix（bHLH） gene,was constructed in an adenoviral vector and then infected into the 3rd passage BMSCs.An inverted fluorescence microscope was used to observe the morphological changes of the infected cells.The expressions of Math5,the neuromarkers neuron-specific enolase（NSE）,neurofilament（NF）,Thy1.1,and the RGC-related genes GAP-43 and Brn3b were examined by Western blot and reverse transcription-polymerase chain reaction（RT-PCR）.The results show that cells infected with Math5 adenoviral vector were able to stably express Math5 and presented with a typical morphology of RGCs.Moreover,these cells expressed NSE,NF,Thy1.1,and GAP-43.Under the synergistic induction conditions of retinal conditioned differentiation medium in combination with epidermal growth factor（EGF） and basic fibroblast growth factor（BFGF）,BMSCs infected with Math5 adenoviral vector had a more typical morphology of RGCs,with a greater number of longer axons that connected with each other and formed a net.In addition,the number of NF positive cells was higher,the expression of Brn3b was detected,and the expressions of NSE,NF,and GAP-43 were significantly up-regulated compared to those of them in the control.These results indicate that BMSCs infected with Math5 are able to differentiate into retinal ganglion-like cells.Moreover,Math5 is a stronger activator of the downstream gene Brn3b than the cytokine,which suggests that it is possible to regulate the survival and axon path determination of these differentiated cells.

Occurrence of MYD88L265P and CD79B mutations in diffuse large b cell lymphoma with bone marrow infiltration:A case report

BACKGROUNDOver the past 20 years,we have gained a deep understanding of the biologicalheterogeneity of diffuse large B cell lymphoma(DLBCL)and have developed arange of new treatment programs based on the characteristics of the disease,bringing us to the era of immune-chemotherapy.However,the effectiveness andmolecular mechanisms of targeted-immunotherapy remain unclear in DLBCL.Targeted-immunotherapy may be beneficial for specific subgroups of patients,thus requiring biomarker assessment.CASE SUMMARYHere,we report a case of MCD subtype DLBCL with MYD88L265P and CD79Bmutations,considered in the initial stage as lymphoplasmic lymphoma(LPL)orWaldenstrom macroglobulinemia(WM).Flow cytometry supported this view;however,the immunohistochemical results of the lymph nodes overturned theabove diagnosis,and the patient was eventually diagnosed with MCD subtypeDLBCL.The presence of a monoclonal IgM component in the serum and infiltrationof small lymphocytes with a phenotype compatible with WM into the bonemarrow led us to propose a hypothesis that the case we report may have transformedfrom LPL/WM.CONCLUSIONThis highlights the possible transformation from WM to DLBCL,CD79B mutationmay be a potential biomarker for predicting this conversion.

α1-antitrypsin combined with bone marrow mesenchymal stem cells regulates retinopathy in diabetic rats via p38 MAPK/NF-κB signaling pathway

Objective:To investigate the effect ofα1-antitrypsin combined with bone marrow mesenchymal stem cells on retinopathy in diabetic rats and its mechanism.Methods:A model of diabetic retinopathy was established by intraperitoneal injection of streptozotocin.The 30 Wistar rats successfully modeled were randomly divided into a model group,a bone marrow mesenchymal stem cell group and a combined group(α1-antitrypsin combined with bone marrow Mesenchymal stem cells),the blood glucose and serum insulin levels of diabetic rats were measured 4 weeks after treatment.Enzyme-linked immunosorbent assay(ELISA)for measuring serum inflammatory factors IL-1β,IL-6 and TNF-α in rats.Observing the pathological morphology of rat retina under hematoxylin-eosin staining(HE).TUNEL staining to observe the apoptosis of rat retinal nerve cells.Immunohistochemical method to detect the expression level of CD45 in retinal tissue.Real-time fluorescence quantitative PCR was used to detect the expression of retinal vascular endothelial growth factor(VEGF),hypoxiainducible factor-1α(HIF-1α),and angiotensinⅡ(ANGⅡ)mRNA.Western blot was used to detect the expression of p38 MAPK/NF-κB signaling pathway-related proteins in the retinal tissue of each group of rats.Results:Compared with the control group,the rats in the model group had increased blood glucose,decreased insulin levels,increased serum IL-1β,IL-6,and TNF-α levels,and had obvious lesions in the retina.CD45 showed high expression in retinal tissue,VEGF,HIF-1α,ANGⅡ mRNA expression increased,p-p38,p-p65,p-IκBα protein expression increased(P<0.05).Compared with the model group,the bone marrow mesenchymal stem cell group and the combined group have decreased blood glucose,increased insulin levels,and decreased serum IL-1β,IL-6 and TNF-α levels.Retinopathy is improved,apoptosis of retinal nerve cells is reduced,CD45 expression in retinal tissue is reduced,VEGF,HIF-1α,ANGⅡ mRNA expression is decreased,and p-p38,p-p65,p-IκBα protein expression is decreased.Compared with the bone marrow mesenchymal stem cell group,the effect of the combined group was more obvious(P<0.05).Conclusion:α1-antitrypsin combined with bone marrow mesenchymal stem cell transplantation can improve the degree of retinopathy in diabetic rats.The mechanism may be related to the inhibition of p38 MAPK/NF-κB signaling pathway.

Association of Morphology and Immunophenotype in Diffuse Large B-Cell Lymphomas with Bone Marrow Infiltration in a Sample Mexican Population

Introduction: Diffuse large B-cell lymphoma (DLBCL), not otherwise specified, is a large B-cell lymphoma with a diffuse growth pattern and aggressive clinical course. It is divided in subgroups according to its morphology, immunophenotype, and primary site. Dissemination to bone marrow occurs in 11% to 35% of cases and can be of concordant or discordant morphology. Objective: To examine the association, the type of bone marrow involvement in relation to the primary site, morphology, immunohistochemistry of DLBCLs and to determine the cases of Epstein-Barr virus positive DLBCLs. Materials and Methods: We reviewed lymph node and extranodal biopsies as well as the respective bone marrow biopsies in all cases of DLBCL diagnosed in the Hospital General de México during the period from 2002 to 2010. We used immunohystochemistry for immunophenotype identification (Hans’s algorithm) and an in-situ hybridization technique to detect presence of Epstein Barr encoded RNA (EBER). Results: We included 108 patients with a mean age of 51.9 years, 59 (55%) were men. DLBCL involved lymph nodes in 60% of cases and palatine tonsils in 13%. The centroblastic variant predominated (80%) and 58% originated from activated B-cells. Infiltration of bone marrow was present in 30% of cases and was discordant in 55% of these cases. Correlation between morphology and bone marrow infiltration was statistically significant (P = 0.0003). Presence of Epstein-Barr virus was demonstrated in 15% of patients older than 50 years. Conclusions: Dissemination to bone marrow occurred in 30% of cases and discordant involvement was most common. DLBCL originating from activated B-lymphocytes predominated and the most common extranodal sites were palatine tonsils, suggesting that our population has a clinical behavior similar to Asiatic populations.

基线PET/CT显像骨髓^(18)F-FDG摄取模式预测DLBCL预后的价值

目的:探讨弥漫大B细胞淋巴瘤(DLBCL)治疗前^(18)F-脱氧葡萄糖(^(18)F-FDG)PET/CT显像中骨髓摄取模式对预测DLBCL患者预后的价值。方法:回顾性分析156例DLBCL患者的临床资料,所有患者治疗前均行骨髓活检、骨髓涂片、流式细胞分析和^(18)F-FDG PET/CT扫描。以正常肝脏^(18)F-FDG摄取为标准,将患者骨髓摄取模式分为骨髓摄取局灶型增高(f PET+)、骨髓摄取弥漫型增高(d PET+)及骨髓摄取正常型(n PET)。生存分析采用Kaplan-Meier法,组间差异比较采用log-rank检验,采用多因素Cox回归分析确定与预后相关的危险因素。结果:156例患者中,f PET+17例,d PET+28例,n PET 111例。临床诊断骨髓浸润阳性21例,阴性135例。复发进展62例,死亡18例。单因素分析显示,Ann Arbor分期Ⅲ/Ⅳ期、B症状、NCCN-IPI评分、乳酸脱氢酶、骨髓浸润及f PET+与患者无进展生存期(PFS)有关(均P<0.05);Ann Arbor分期Ⅲ/Ⅳ期、NCCN-IPI评分、乳酸脱氢酶、骨髓浸润及f PET+与患者总生存期(OS)有关(均P<0.05);多因素分析显示,Ann Arbor分期III/IV期、乳酸脱氢酶和f PET+(均P<0.05)是PFS独立预测因子。在OS的多变量分析中没有独立预测因子。结论:DLBCL患者治疗前^(18)F-FDG显像中骨髓摄取模式对预后有预测价值,f PET+是患者PFS的独立危险因素。

尿石素B对骨髓来源巨噬细胞向破骨细胞分化的调控机制

背景:尿石素B在机体免疫应答中起重要调节作用,具备抗炎、抗氧化和抗癌的特性,并能抑制Raw 264.7细胞向破骨细胞分化,但其对于骨髓来源的巨噬细胞向破骨细胞分化的具体作用及机制尚未阐明。系统性研究破骨细胞过度活化的调控机制,有助于探索新的治疗靶点,筛选研发更安全、有效的治疗药物,为阻断破骨细胞过度活化提供新思路。目的:利用骨髓来源的巨噬细胞建立体外破骨细胞分化模型,探究尿石素B对核因子κB受体活化因子配体介导破骨细胞分化的影响,并系统性分析其作用机制。方法:(1)采用CCK-8法筛选尿石素B干预骨髓来源巨噬细胞的安全工作浓度;(2)用不同浓度(0,12.5,25,50μmol/L)尿石素B干预骨髓来源的巨噬细胞向破骨细胞分化,进行抗酒石酸酸性磷酸酶染色观察破骨细胞的数目及面积大小;(3)不同浓度(0,12.5,25,50μmol/L)尿石素B干预骨髓来源巨噬细胞的破骨分化,通过实时荧光定量PCR检测破骨特异性基因的相对表达水平;(4)蛋白印迹实验观察尿石素B对骨髓来源巨噬细胞P65、ERK信号通路的影响;(5)蛋白印迹实验检测尿石素B对骨髓来源巨噬细胞的破骨分化关键转录因子活化T细胞核因子1和c-Fos的影响。结果与结论:(1)50μmol/L及以下浓度的尿石素B对骨髓来源巨噬细胞的增殖无影响,能显著抑制骨髓来源巨噬细胞的破骨分化;(2)尿石素B主要在破骨形成前中期抑制骨髓来源巨噬细胞的破骨分化;(3)尿石素B可下调骨髓来源巨噬细胞中破骨特异性基因的相对表达水平;(4)50μmol/L的尿石素B抑制骨髓来源巨噬细胞的P65和ERK磷酸化水平,进而抑制破骨细胞形成;(5)50μmol/L的尿石素B抑制骨髓来源的巨噬细胞中破骨分化关键转录因子活化T细胞核因子1和c-Fos的表达;(6)提示尿石素B通过P65/ERK信号轴下调破骨关键转录因子活化T细胞核因子1、c-Fos的表达,抑制下游破骨特异性基因的表达,从而抑制骨髓来源的巨噬细胞向破骨细胞分化。

BACE-1修饰的人骨髓间充质干细胞对创伤性颅脑损伤大鼠脑组织的保护作用

目的探究β-位点淀粉样前体蛋白剪切酶-1(BACE-1)修饰的人骨髓间充质干细胞(BMSCs)对创伤性颅脑损伤(TBI)大鼠脑组织的保护作用。方法将敲低BACE-1基因的腺病毒及空载体腺病毒感染BMSCs,并检测绿色荧光和BACE-1表达。将100只大鼠随机分为假手术(Sham)组、TBI组、空载体腺病毒感染BMSCs(Ad-BMSCs)组和敲低BACE-1基因的腺病毒感染BMSCs(Ad-si-BACE-1-BMSCs)组,每组各25只。采用Marmarou′s自由落体方法建立大鼠TBI模型,Sham组仅切开、缝合头皮,不致伤。建模2 h后,Ad-si-BACE-1-BMSCs组和Ad-BMSCs组分别经尾静脉注射敲低BACE-1基因的腺病毒及空载体腺病毒感染的BMSCs,Sham组和TBI组均给予等体积生理盐水。BMSCs移植7 d后,Morris水迷宫实验检测大鼠认知能力;苏木精-伊红染色和尼氏染色评估大鼠海马组织损伤;TUNEL染色检测海马神经元凋亡;硫黄素-S染色、免疫组化染色检测海马组织β-淀粉样蛋白(Aβ)含量;硫代巴比妥酸法和全自动生化分析仪检测海马组织丙二醛(MDA)、超氧化物歧化酶(SOD)水平;免疫荧光染色检测海马组织离子钙结合接头分子1(Iba-1)^(+)肿瘤坏死因子-α(TNF-α)+、Iba-1^(+)白细胞介素(IL)-6^(+)、Iba-1^(+)IL-1β^(+)、胶质纤维酸性蛋白(GFAP)+TNF-α^(+)、GFAP+IL-6^(+)、GFAP+IL-1β^(+)水平;蛋白免疫印迹(Western blot)检测海马组织BACE-1、Aβ、TNF-α、IL-6、IL-1β水平。结果与Sham组比较,TBI组大鼠逃避潜伏期增加、到达先前平台的次数和在平台停留的时间减少,海马神经元排列紊乱,尼氏小体减少,TUNEL阳性率增加,海马组织Aβ、BACE-1、TNF-α、IL-6、IL-1β蛋白、MDA含量、Iba-1^(+)TNF-α^(+)、Iba-1^(+)IL-6^(+)、Iba-1^(+)IL-1β^(+)、GFAP+TNF-α^(+)、GFAP+IL-6^(+)、GFAP+IL-1β^(+)细胞数增加,SOD含量减少(P均<0.05);与TBI组比较,Ad-BMSCs组和Ad-si-BACE-1-BMSCs组大鼠逃避潜伏期减少、到达先前平台的次数和在平台停留的时间增加,神经元排列较规则,尼氏小体增加,TUNEL阳性率减少,海马组织Aβ、BACE-1、TNF-α、IL-6、IL-1β蛋白、MDA含量、Iba-1^(+)TNF-α^(+)、Iba-1^(+)IL-6^(+)、Iba-1^(+)IL-1β^(+)、GFAP+TNF-α^(+)、GFAP+IL-6^(+)、GFAP+IL-1β^(+)细胞数减少,SOD含量增加(P均<0.05);且Ad-si-BACE-1-BMSCs对大鼠上述指标的影响优于Ad-BMSCs(P<0.05)。结论BACE-1修饰的人BMSCs能够抑制TBI大鼠氧化应激和炎症反应,对TBI大鼠脑组织具有保护作用。

血清游离轻链在B细胞非霍奇金淋巴瘤中的表达水平及诊断价值

目的:探讨血清游离轻链(sFLC)在B细胞非霍奇金淋巴瘤(B-NHL)中的表达水平及诊断价值。方法:回顾性分析2014年1月至2021年12月江苏省人民医院394例初治B-NHL患者的血清游离轻链结果,比较不同亚型B-NHL中sFLC的分泌水平。评估sFLC分泌水平在华氏巨球蛋白(WM)诊断中的价值。结果:sFLC增高比例、sFLC(κ/λ)异常比例及sFLC(κ+λ)分泌水平在不同B-NHL亚型中均存在差异性,WM的sFLC增高者比例(82.6%)与sFLC(κ/λ)比值异常患者比例(87.0%)最高,而sFLC增高者比例最低的为FL(18.0%)与DLBCL(12.8%)(P<0.05)。sFLC的表达水平可以辅助诊断WM(AUC=0.874,P<0.001,0.779-0.970)。同时,较高的sFLC水平及sFLC克隆模式可以预测淋巴瘤骨髓浸润的可能。结论:B-NHL患者血清中存在游离轻链,轻链的增高水平和增高类型与淋巴瘤类别存在相关性,且骨髓浸润患者有着更高的sFLC(κ+λ)表达水平。

补阳还五汤通过调控PI3K/Akt、JAK2/STAT3信号促进BMSC趋化迁移对外伤性脊髓损伤大鼠神经元活性及认知功能的影响

目的研究补阳还五汤通过调控磷脂酰肌醇-3激酶/蛋白激酶B(PI3K/Akt)、内源性酪氨酸激酶(JAK)2/信号传导和转录启动因子(STAT)3信号促进骨髓间充质干细胞(BMSCs)趋化迁移对外伤性脊髓损伤大鼠的神经元活性及认知功能的影响。方法选取健康大鼠53只,随机分为健康组(健康大鼠常规饲养)、损伤组(建立脊髓损伤模型)、干预组(补阳还五汤治疗)、对照组(甲泼尼龙治疗),每组12只,剩余5只大鼠用于补阳还五汤含药血清制备。流式细胞术鉴定BMSCs细胞。Transwell小室法测大鼠BMSCs迁移。高架十字迷宫和Morris水迷宫实验检测大鼠认知功能。苏木素-伊红(HE)染色检测脊髓组织病理形态。TUNEL测脊髓组织神经细胞凋亡。免疫组化检测p-JAK2、p-STAT3。Western印迹测PI3K、p-PI3K、Akt、p-Akt。结果传代后的培养细胞呈旋窝状或放射状贴壁生长,细胞多呈星形、梭形或三角状,培养3代后,细胞贴壁加快、形态均一,呈旋窝状或单层放射状生长。培养细胞表面抗原CD29、CD90为阳性,CD31、CD45为阴性,提示其为BMSCs细胞。与健康组相比,损伤组总路程、进入开臂次数、穿越平台次数显著降低,不同时间的潜伏期显著升高(P<0.05)。与损伤组相比,干预组与对照组总路程、进入开臂次数、穿越平台次数显著升高,不同时间的潜伏期显著降低(P<0.05)。干预组与对照组各指标对比无统计学差异(P>0.05)。健康组脊髓组织结构完整。损伤组脊髓组织疏松水肿,有细胞空泡变性产生。相较于损伤组,干预组与对照组大鼠脊髓组织病理形态有所改善。与健康组相比,损伤组BMSCs、PI3K、Akt、p-PI3K、p-Akt显著降低,神经细胞凋亡率、p-JAK2、p-STAT3显著升高(P<0.05)。与损伤组相比,干预组BMSCs、PI3K、Akt、p-PI3K、p-Akt显著升高,神经细胞凋亡率、p-JAK2、p-STAT3显著降低(P<0.05)。干预组与对照组各指标水平无统计学差异(P>0.05)。结论补阳还五汤通过激活PI3K/Akt通路抑制JAK2/STAT3信号通路的激活,促进BMSCs的迁移,减轻神经细胞的凋亡,起到神经保护的作用,从而改善脊髓损伤大鼠的认知功能。

18F-FDG PET/CT对初诊弥漫大B细胞淋巴瘤骨髓浸润的诊断及预后评估价值

目的:评价18F-FDG PET/CT对初诊弥漫大B细胞淋巴瘤(DLBCL)骨髓浸润的诊断价值,并与骨髓活检(BMB)结果进行比较,探讨18F-FDG PET/CT诊断的骨髓浸润及其他因素是否有独立的预后预测价值。方法:纳入94例在南京医科大学附属上海一院临床医学院行PET/CT检查的初诊DLBCL患者,PET/CT检查前后2周内行骨髓活检,检查后行规范化治疗。记录各病例的PET/CT骨髓摄取表现。骨髓浸润的诊断标准为BMB阳性或影像学随访证实有PET/CT局灶性骨髓浸润灶。比较PET/CT骨髓不同摄取表现的患者临床资料差异并对照分析PET/CT与BMB诊断骨髓浸润的价值。Kaplan-Meier法绘制生存曲线分析患者组间无进展生存期(PFS)差异,log-rank检验比较组间PFS率差异,COX回归模型分析影响PFS的独立危险因素。结果:94例DLBCL患者中,18F-FDG PET/CT表现为局灶性骨髓摄取(f PET)34例,超级骨髓摄取(s BMU)7例,高于肝实质的弥漫均匀性摄取(d PET)11例,其余42例骨髓摄取未见异常(n PET)。n PET组BMB均阴性,s BMU组BMB均阳性,而d PET、f PET组的BMB阳性率分别为27.3%(3/11)和20.6%(7/34)。d PET组的血红蛋白水平及白细胞数均显著低于n PET组(P<0.001,P=0.026)。与f PET组比较,s BMU组更易伴B症状及乳酸脱氢酶水平升高。全部患者共诊断骨髓浸润44例,其中BMB阳性17例,BMB诊断骨髓浸润的灵敏度、特异性分别为38.6%(17/44)、100%(50/50)。将f PET及s BMU作为PET骨髓浸润阳性标准,PET/CT诊断骨髓浸润的灵敏度、特异性分别为93.2%(41/44)、100%(50/50)。Kaplan-Meier分析结果显示,n PET与d PET组的2年PFS率无明显差异(P>0.05),而s BMU组的2年PFS率明显低于f PET组(P<0.001)。多因素分析显示,高分期(HR=9.010,P=0.04)及s BMU(HR=3.964,P=0.002)是影响PFS的独立危险因素。结论:DLBCL的18F-FDG PET/CT骨髓摄取增高可表现为d PET、f PET和s BMU,其中f PET和s BMU可取代骨髓活检诊断骨髓浸润,d PET虽然不完全除外骨髓浸润可能,但不影响预后,因此可诊断为骨髓浸润阴性。sBMU为独立的预后危险因素。

Clinical significance of HBME-1,Galectin-3,and CK19 expression and the status of BRAF mutation in papillary thyroid carcinoma

Objective The aim of this study was to explore the clinical significance of the expression of proteins human bone marrow endothelial cell markers(HBME-1), Galectin-3, and cytokeratin19(CK19), as well as the status of v-raf murine sarcoma viral oncogene homolog B1(BRAF) mutation in papillary thyroid carcinoma(PTC). Methods Immunohistochemical staining was performed in 82 specimens each of PTC and papillary benign lesions to detect the expression of HBME-1, Galectin-3, and CK19. Polymerase chain reaction(PCR) and gene sequencing were performed on 60 specimens each of PTC and papillary benign lesions to detect the status of BRAF mutation. Results The positive expression ratios of HBME-1, Galectin-3, and CK19 in PTC were 98.8%, 97.6% and 100% respectively, which were significantly higher than the expressions in papillary benign lesions(P < 0.05). No significant relationship was observed between the expression of these makers and the clinicopathological features of PTC. The sensitivity of co-expression of HBME-1 and CK19 or HBME-1 and Galectin-3 as diagnostic criteria of PTC was 99.9%, with a specificity of 95.4%. BRAF mutation was detected in 40 of 60 PTC(66.7%) specimens. There was a statistical difference in BRAF mutations between PTC and papillary benign lesions(P < 0.05); there were no associations between BRAF mutation and the clinicopathological features of PTC. Conclusion Combined immunohistochemical staining of HBME-1, Galectin-3, and CK19 can further improve the sensitivity and specificity of differential diagnosis of PTC. BRAF mutation is a significant genetic event, which may have diagnostic value for PTC.

基于多模态图像构建CNN-ViT模型在弥漫性大B细胞淋巴瘤骨髓受累诊断中的应用

目的设计一种融合多模态图像深度学习模型CNN-ViT,诊断弥漫性大B细胞淋巴瘤(DLBCL)骨髓受累。资料与方法回顾性收集2012年11月—2022年6月福建省立医院经病理证实的DLBCL 78例,其中无骨髓受累46例,有骨髓受累32例,所有患者在化疗前均行全身18F-FDG PET/CT检查、骨髓穿刺细胞涂片和(或)骨髓活检。选取骨盆区域PET及CT图像共9828张。将上述数据按7∶1∶2随机分为训练集6858张、验证集982张和测试集1988张。结合传统的卷积神经网络(CNN)和Vision-Transformer(ViT)模型设计CNN-ViT模型,分别提取PET和CT图像特征,预测骨髓受累情况。使用测试集的混淆矩阵和损失函数的变化、准确度、敏感度、特异度和F1_score评价模型的性能。结果CNN-ViT模型诊断DLBCL骨髓受累的准确度、特异度、敏感度和F1_score分别为0.988、0.971、0.997、0.987。结论CNN-ViT模型可以准确评估DLBCL骨髓受累情况。

骨髓涂片形态对弥漫性大B细胞淋巴瘤骨髓侵犯的诊断价值

目的探讨骨髓涂片形态对弥漫性大B细胞淋巴瘤(DLBCL)骨髓侵犯的诊断价值。方法回顾性分析2010年1月至2021年4月本院收治的2100例DLBCL患者的临床资料,观察患者骨髓涂片形态学,比较骨髓活检与实验室检测结果及DLBCL骨髓侵犯涂片和活检的一致性。结果骨髓活检显示,发生骨髓侵犯患者235例,其中包括结旁型患者125例,散在分布型患者100例及混合型患者10例。患者Ki-67数值为3%~88%,平均为69%,其中Ki-67数值≥80%患者40例;骨髓涂片显示,骨髓侵犯患者210例,两种检查方式结果统计后,245例患者骨髓侵犯,两种检查方式用于DLBCL骨髓浸润诊断的一致性良好(Kappa=0.905);DLBCL细胞大小适中,细胞核呈圆形或不规则型,染色质颜色表现为深紫红色,核仁数量大多为1~5个;胞浆为蓝色。结论DLBCL骨髓侵犯诊断中骨髓涂片形态检查可作为有效方法,当有核细胞中不典型淋巴细胞占比超过1%,则需进一步诊断是否发生骨髓侵犯。

Bone marrow-derived mesenchymal stem cells modulate autophagy in RAW264.7 macrophages via the phosphoinositide 3-kinase/protein kinase B/heme oxygenase-1 signaling pathway under oxygen-glucose deprivation/restoration conditions

Background: Autophagy of alveolar macrophages is a crucial process in ischemia/reperfusion injury-induced acute lung injury (ALI). Bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent cells with the potential for repairing injured sites and regulating autophagy. This study was to investigate the influence of BM-MSCs on autophagy of macrophages in the oxygen-glucose deprivation/restoration (OGD/R) microenvironment and to explore the potential mechanism.Methods: We established a co-culture system of macrophages (RAW264.7) with BM-MSCs under OGD/R conditionsin vitro. RAW264.7 cells were transfected with recombinant adenovirus (Ad-mCherry-GFP-LC3B) and autophagic status of RAW264.7 cells was observed under a fluorescence microscope. Autophagy-related proteins light chain 3 (LC3)-I, LC3-II, and p62 in RAW264.7 cells were detected by Western blotting. We used microarray expression analysis to identify the differently expressed genes between OGD/R treated macrophages and macrophages co-culture with BM-MSCs. We investigated the gene heme oxygenase-1 (HO-1), which is downstream of the phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) signaling pathway.Results: The ratio of LC3-II/LC3-I of OGD/R treated RAW264.7 cells was increased (1.27 ± 0.20vs. 0.44 ± 0.08,t = 6.67,P < 0.05), while the expression of p62 was decreased (0.77 ± 0.04vs. 0.95 ± 0.10,t = 2.90,P < 0.05), and PI3K (0.40 ± 0.06vs. 0.63 ± 0.10,t = 3.42,P < 0.05) and p-Akt/Akt ratio was also decreased (0.39 ± 0.02vs. 0.58 ± 0.03,t = 9.13,P < 0.05). BM-MSCs reduced the LC3-II/LC3-I ratio of OGD/R treated RAW264.7 cells (0.68 ± 0.14vs. 1.27 ± 0.20,t = 4.12,P < 0.05), up-regulated p62 expression (1.10 ± 0.20vs. 0.77 ± 0.04,t = 2.80,P < 0.05), and up-regulated PI3K (0.54 ± 0.05vs. 0.40 ± 0.06,t = 3.11,P < 0.05) and p-Akt/Akt ratios (0.52 ± 0.05vs. 0.39 ± 0.02,t = 9.13,P < 0.05). A whole-genome microarray assay screened the differentially expressed geneHO-1, which is downstream of the PI3K/Akt signaling pathway, and the alteration ofHO-1 mRNA and protein expression was consistent with the data on PI3K/Akt pathway.Conclusions: Our results suggest the existence of the PI3K/Akt/HO-1 signaling pathway in RAW264.7 cells under OGD/R circumstancesin vitro, revealing the mechanism underlying BM-MSC-mediated regulation of autophagy and enriching the understanding of potential therapeutic targets for the treatment of ALI.