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Background Preoperative prediction of microvascular invasion (MVI) in hepatocellular carcinoma (HCC) may optimize individualized treatment decision-making. This study aimed to investigate the prognostic differences between HCC patients undergoing liver resection (LR) and liver transplantation (LT) based on predicted MVI risks. Methods We analysed 905 patients who underwent LR, including 524 who underwent anatomical resection (AR) and 117 who underwent LT for HCC within the Milan criteria using propensity score matching. A nomogram model was used to predict preoperative MVI risk. Results The concordance indices of the nomogram for predicting MVI were 0.809 and 0.838 in patients undergoing LR and LT, respectively. Based on an optimal cut-off value of 200 points, the nomogram defined patients as high- or low-risk MVI groups. LT resulted in a lower 5-year recurrence rate and higher 5-year overall survival (OS) rate than LR among the high-risk patients (23.6% vs 73.2%, P  <   0.001; 87.8% vs 48.1%, P  <   0.001) and low-risk patients (19.0% vs 45.7%, P  <   0.001; 86.5% vs 70.0%, P  =   0.002). The hazard ratios (HRs) of LT vs LR for recurrence and OS were 0.18 (95% confidence interval [CI], 0.09–0.37) and 0.12 (95% CI, 0.04–0.37) among the high-risk patients and 0.37 (95% CI, 0.21–0.66) and 0.36 (95% CI, 0.17–0.78) among the low-risk patients. LT also provided a lower 5-year recurrence rate and higher 5-year OS rate than AR among the high-risk patients (24.8% vs 63.5%, P  =   0.001; 86.7% vs 65.7%, P  =   0.004), with HRs of LT vs AR for recurrence and OS being 0.24 (95% CI, 0.11–0.53) and 0.17 (95% CI, 0.06–0.52), respectively. The 5-year recurrence and OS rates between patients undergoing LT and AR were not significantly different in the low-risk patients (19.4% vs 28.3%, P  =   0.129; 85.7% vs 77.8%, P  =   0.161). Conclusions LT was superior to LR for patients with HCC within the Milan criteria with a predicted high or low risk of MVI. No significant differences in prognosis were found between LT and AR in patients with a low risk of MVI. [ABSTRACT FROM AUTHOR]

Background: Axl is a receptor tyrosine kinase which plays an important role in multiple human malignancies. Design: The Axl expression was examined in several hepatocellular carcinoma(HCC) cell lines, paired tumor and nontumorous samples. Then, we examined cell growth curve, cell apoptosis and cell migration in SMMC-7721 cells over-expressed with Axl or siRNA against Axl, respectively. Finally, the prognostic value of Axl was investigated in a prospective cohort of 246 consecutive HCC patients undergoing curative hepatoectomy. Results: We found Axl was positive in 22% of examined tumor tissues and all four cell lines. Over-expressing Axl in SMMC-7721 cells accelerated cell growth, cell migration and inhibited cell apoptosis, while knock-down of Axl exerted opposite effect. Axl expression was closely associated with serum AFP, multiple tumors, absence of encapsulation, microvascular invasion, and advanced BCLC or TNM stage. Patients with positive Axl staining had a higher 5-year recurrence rate (92% vs. 71%, P<0.001) and a lower 5-year survival rate (9% vs. 48%, P<0.001) than those with negative staining. The multivariate analyses showed that Axl expression was an independent factor for both tumor recurrence (HR: 1.725; 95% CI: 1.219–2.441) and survival (1.847; 1.291–2.642). Conclusion: Axl expression suggests more aggressive tumor invasiveness and predicts worse prognosis for HCC patients undergoing resection. [ABSTRACT FROM AUTHOR]

Background. The effectiveness of adjuvant transarterial chemoembolization (TACE) for intrahepatic cholangiocarcinoma (ICC) after hepatectomy remains unclear. This study was performed to identify ICC patients who would benefit from adjuvant TACE. Patients and Methods. The study included 553 patients who underwent hepatectomy for ICC between January 2008 and February 2011 at the Eastern Hepatobiliary Surgery Hospital and who were treated with or without TACE (122 with TACE and 431 without TACE). Survival risk stratification was performed using the established prognostic nomogram (ICC nomogram). The predictive performance was evaluated by concordance index and calibration. The tumor recurrence and overall survival (OS) rates were analyzed by the Kaplan-Meier method before and after propensity score matching (PSM). Results. The predictive performance of the ICC nomogram was demonstrated by the well-fitted calibration curves and an optimal c-index of 0.71 for OS prediction. In the whole cohort, the 5-year recurrence and OS rates between the TACE and non-TACE groups were significantly different (5-year recurrence: 72.9% vs. 78.1%; OS: 38.4% vs. 29.7%). After 1:1 PSM, the TACE and non-TACE groups (122 patients each) had similar 5-year recurrence and OS rates (5-year recurrence: 72.9% vs. 74.2%; OS: 38.4% vs. 36.0%). By survival risk stratification based on ICC nomogram, only the patients in the lowest tertile (nomogram scores ≥77) benefited from adjuvant TACE (TACE vs. non-TACE groups: 90.4% vs. 95.9% for 5-year recurrence; 21.3% vs. 6.2% for 5-year OS). Conclusion. Adjuvant TACE following liver resection might be suitable for ICC patients with high ICC nomogram scores (≥77). [ABSTRACT FROM AUTHOR]

Most previous studies focused on a small number of heat shock proteins (Hsps) and their relationships with embryogenesis, and the actual roles of these Hsps in normal and abnormal embryonic development remain unclear. It was found in the present systemic study that except for Grp170, whose expression was not detectable at GD18, all 19 Hsps of Hsp70, Hsp90 and Hsp110 families were expressed in the normal development of embryonic palate tissue in mice, but their expression patterns varied with different Hsps, presenting as a correlation with the developmental phases. In the treatment group by all-trans retinoic acid (atRA), the messenger RNA (mRNA) abundance of HspA1A, HspA1L, HspA8, HspA9, HspA12A, HspA12B, HspA13, HspA14, Hsp90AA1, Hsp90AB1, Grp94, Trap1, Hsp105, Hsp110 and Grp170 was higher in the palates at GD11 (the beginning of palate development), the mRNA abundance of HspA1A, HspA12A and HspA12B was higher at GD18 (before birth) and an mRNA expression peak of HspA1L, HspA8, HspA9, Hsp90AA1, Grp94, Hsp110 and Grp170 was observed at GD17. The mRNA abundance of most genes in atRA-induced cleft palates of the treatment group was different from that of the control group. Grp78, HspA14 and Hsp105 were closely associated with the normal palate development and cleft palate in mouse embryo, possibly as palate development-related genes. Except Grp170, the other genes may be closely associated with the development of mouse palates through participating in the stress response process and/or the antiapoptosis process. [ABSTRACT FROM PUBLISHER]

Heat shock proteins (Hsps) are thought of as chaperones of morphologic development of cells and organisms and are believed to be closely related to normal and abnormal embryonic development. It was found, in the present study, that 19 Hsps of the three Hsp70, Hsp90, and Hsp110 families were expressed in embryonic forelimb tissue of normal mice, but in different patterns, showing a characteristic correlation with the developmental phases. The tendency that the expression of many genes changed with embryonic age, increasing in the all-trans retinoic acid (atRA)-induced limb malformation groups, was similar to that of the control group, but messenger RNA abundance of most of these genes was significantly different from that in the control group. HspA12B, HspA14, Trap1, and Hsp105 may be limb-development-related genes; Grp78 may play an important role in limb development. [ABSTRACT FROM AUTHOR]

Background. Postoperative extrahepatic metastasis (EHM) contributes to a poor prognosis in patients with hepatocellular carcinoma (HCC) after hepatectomy. This study was aimed to develop a practical method that can be used to predict postoperative EHM. Methods. In total, 578 patients were enrolled. We analyzed the clinicopathological features of the tumors and did a long-term follow-up to observe HCC recurrence. Postoperative EHM was detected in 136 patients, and multivariate analysis was used to confirm independent risk factors for postoperative EHM. After the factors were identified, a predictive scoring system was constructed as a weighted sum of these factors. The cutoff value that determines a high risk for EHM was defined by maximizing the Youden's index of the receiver operating characteristic curve. Results. Microvascular invasion, incomplete capsule, and larger tumor diameter were the three independent factors predictive for a high risk for EHM. The scoring system was derived with an area under the curve (AUC) of 0.81 for postoperative 10-year EHM prediction. A cutoff value of 43 was derived and validated with a sensitivity >90% and specificity>60% to predict the development of EHM. This system was further verified in a subgroup of Barcelona Clinic Liver Cancer stage 0-A patients with an AUC of 0.82. When the cutoff value was set at 43, the sensitivity and specificity were 90.38% and 64.88%, respectively. Conclusions. Our predictive scoring system may be used to identify HCC patients who have a high risk for EHM following curative hepatectomy. [ABSTRACT FROM AUTHOR]

Preantral follicle in vitro culture systems have been successfully or nearly successfully established for sheep, pig and mouse, and applied on follicle development and regulation research on reproductive biology and physiology. However, there have been few studies concerning rat preantral follicle in vitro development. The objective is to establish an in vitro culture system for rat preantral follicles which can be used for reproductive biology and toxicology research. Rat preantral follicles are mechanically separated, cultured in vitro in single follicle mode for continuous 12 days using 96-well plates, and then administrated ovulation induction. The observation on follicle development, hormone level, and ovum formation are recorded and assessed. Taking in vivo growth and in vitro maturation of ocytes group as control group, in vitro growth and maturation of oocytes group is assessed to see whether this in vitro culture method is successful. The conditions for rat follicle culture are determined based on the mouse pre-antral follicle culture. The in vitro culture system for rat preantral follicles established in this study is feasible and successful, and can serve as model for reproductive biology and toxicology research. [ABSTRACT FROM AUTHOR]

Fertility and early embryonic developmental toxicity in rats were evaluated by intravenously administering astragaloside IV (AS-IV) daily at 0.25, 0.5, and 1.0 mg/kg for 4 weeks before mating, throughout the mating period, and continuing to day 6 of gestation period in females. Perinatal toxicity in rats was evaluated on gestational days (GD) 15 to 21 and lactational days LD (LD) 1 to 21. Astragaloside IV had no maternal toxicity at 0.25 to 1.0 mg/kg in rats. Although it has an inhibitory effect on female fertility in F0/F1 rats, AS-IV was devoid of early embryonic developmental toxicity in F0/F1 rats and in the survival parameters of F1 postnatal rats. Maternal AS-IV exposure at the dose of 1.0 mg/kg per d resulted in a significant delay in time for fur development, eye opening, and cliff parry reflex of pups compared to control group (P < .05), whereas it did not affect the memory and learning of F1 pups. [ABSTRACT FROM PUBLISHER]

It is reported that salidroside, the main component of a traditional Chinese medicine, Rhodiola rosea, has the efficacy of protecting Coxsackie virus impairment. As part of a safety evaluation on salidroside for use in the treatment of viral myocarditis, the present study evaluated potential genotoxicity of salidroside by using the standard battery of tests (i.e., bacterial reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay) recommended by the State Food and Drug Administration of China. The results showed that salidroside was not genotoxic under the conditions of the reverse mutation assay, chromosomal aberrations assay, and mouse micronucleus assay conditions. The anticipated clinical dose seems to be smaller than the doses administered in the genotoxicity assays. With confirmation from further toxicity studies, salidroside would hopefully prove to be a safe anti-Coxsackie virus agent. [ABSTRACT FROM AUTHOR]

The article presents a study which develops Astragaloside Iv, a natural product purified from Chinese medical herb, as a cardioprotective agent to treat cardiovascular diseases. The study uses astragaloside IV in Sprague-Dawley rats and New Zealand White rabbits during gestation for 6-18 days. It reveals that astragloside IV is maternally toxic at 1.0 mg kg in rats and fetotoxic at a dose more than 0.5 mg kg.

Background & Aims The impact of hepatitis B virus (HBV) infection on outcomes after resection of intrahepatic cholangiocarcinoma (ICC) has not been reported. The aim of this study was to examine the impact of antiviral therapy on survival outcomes after liver resection for patients with ICC and underlying HBV infection. Methods Data on 928 patients with ICC and HBV infection who underwent liver resection at two medical centers between 2006 and 2011 were analyzed. Data on viral reactivation, tumor recurrence, cancer-specific survival (CSS) and overall survival (OS) were obtained. Survival rates were analyzed using the time-dependent Cox regression model adjusted for potential covariates. Results Postoperative viral reactivation occurred in 3.3%, 8.3% and 15.7% of patients who received preoperative antiviral therapy, who did not receive preoperative antiviral therapy with a low, or a high HBV-DNA level (< or ≥2,000 IU/ml), respectively ( p  <0.001). A high viral level and viral reactivation were independent risk factors of recurrence (hazard ratio [HR] 1.22 and 1.34), CSS (HR 1.36 and 1.46) and OS (HR 1.23 and 1.36). Five-year recurrence, CSS and OS were better in patients who received antiviral therapy (70.5%, 46.9% and 43.0%) compared with patients who did not receive antiviral therapy and had a high viral level (86.5%, 20.9% and 20.5%, all p  <0.001), respectively. The differences in recurrence, CSS and OS were minimal compared with no-antiviral therapy patients with a low viral level (71.7%, 35.5% and 33.5%, p  = 0.057, 0.051 and 0.060, respectively). Compared to patients with a high viral level who received no antiviral therapy, patients who initiated antiviral therapy either before or after surgery had better long-term outcomes (HR 0.44 and 0.54 for recurrence; 0.38 and 0.57 for CSS; 0.46 and 0.54 for OS, respectively). Conclusions Viral reactivation was associated with worse prognoses after liver resection for HBV-infected patients with ICC. Antiviral therapy decreased viral reactivation and prolonged long-term survival for patients with ICC and a high viral level. Lay summary Postoperative hepatitis B virus reactivation was associated with an increased complication rate and a decreased survival rate after liver resection in patients with ICC and hepatitis B virus infection. Antiviral therapy before liver resection reduced the risk of postoperative viral reactivation. Both pre- and postoperative antiviral therapy was effective in prolonging patient survival. [ABSTRACT FROM AUTHOR]

Portal vein tumor thrombus (PVTT) is very common and it plays a major role in the prognosis and clinical staging of hepatocellular carcinoma (HCC). We have published the first version of the guideline in 2016 and revised in 2018. Over the past several years, many new evidences for the treatment of PVTT become available, especially for the advent of new targeted drugs and immune checkpoint inhibitors which have further improved the prognosis of PVTT. So, the Chinese Association of Liver Cancer and Chinese Medical Doctor Association revised the 2018 version of the guideline to adapt to the development of PVTT treatment. Future treatment strategies for HCC with PVTT in China would depend on new evidences from more future clinical trials., (Copyright © 2022 by The Author(s). Published by S. Karger AG, Basel.)

The wide spread of coronavirus disease 2019 is currently the most rigorous health threat, and the clinical outcomes of severe patients are extremely poor. In this study, we establish an early warning nomogram model related to severe versus common COVID-19. A total of 1059 COVID-19 patients were analyzed in the primary cohort and divided into common and severe according to the guidelines on the Diagnosis and Treatment of COVID-19 by the National Health Commission of China (7th version). The clinical data were collected for logistic regression analysis to assess the risk factors for severe versus common type. Furthermore, 123 COVID-19 patients were reviewed as the validation cohort to assess the performance of this model. Multivariate logistic analysis revealed that age, dyspnea, lymphocyte count, C-reactive protein and interleukin-6 were independent factors for prewarning the severe type occurrence. Then, the early warning nomogram model including these risk factors for inferring the severe disease occurrence out of common type of COVID-19 was constructed. The C-index of this nomogram in the primary cohort was 0.863, 95% confidence interval (CI) (0.836-0.889). Meanwhile, in the validation cohort, the C-index of this nomogram was 0.889, 95% CI (0.828-0.950). In both the primary cohort and validation cohorts, the calibration curve showed good agreement between prediction and actual probability. The early warning model shows that data at the very beginning including age, dyspnea, lymphocyte count, CRP, and IL-6 may prewarn the severe disease occurrence to some extent, which could help clinicians early and timely treatment.

Over-expression of aspartyl (asparagynal)-β-hydroxylase (ASPH) contributes to hepatocellular carcinoma (HCC) invasiveness, but the role of ASPH hydroxylase activity in this process remains to be defined. As such, the current study investigated the role of ASPH hydroxylase activity in downstream signalling of HCC tumorgenesis and, specifically, metastasis development. Over-expression of wild-type ASPH, but not a hydroxylase mutant, promoted HCC cell migration in vitro, as well as intrahepatic and distant metastases in vivo. The enhanced migration and epithelial to mesenchymal transition (EMT) activation was notably absent in response to hydroxylase activity blockade. Vimentin, a regulator of EMT, interacted with ASPH and likely mediated the effect of ASPH hydroxylase activity with cell migration. The enhanced hydroxylase activity in tumor tissues predicted worse prognoses of HCC patients. Collectively, the hydroxylase activity of ASPH affected HCC metastasis through interacting with vimentin and regulating EMT. As such, ASPH might be a promising therapeutic target of HCC., (Copyright © 2018. Published by Elsevier B.V.)

The aim of the present study was to compare the effectiveness of transarterial chemoembolization (TACE), TACE combined with Jie-du granules (JD), and TACE combined with sorafenib (SOR) for treating patients with unresectable hepatocellular carcinoma (HCC). For this purpose, we conducted a retrospective analysis of data from 266 consecutive patients with unresectable HCC who underwent TACE treatment at the Shanghai Hospital and Eastern Hepatic Surgery Hospital between Jan 2009 and Dec 2010. We prospectively analyzed patient survival and progression times as well as independent predictors, within a follow-up period of 86 months. Patients were divided into TACE-JD (n = 75), TACE-SOR (n = 124) and TACE (n = 67) groups. Median overall survival (OS) times being: TACE-JD, 21.43 months; TACE-SOR, 23.23 months; TACE, 13.97 months (TACE-SOR vs TACE, P < 0.001; TACE-SOR vs TACE-JD, P = 0.852; TACE-JD vs TACE, P < 0.001). The median times to progression (TTP) were as follows: TACE-JD, 8.67 months; TACE-SOR, 5.37 months; TACE, 4.57 months (TACE-SOR vs TACE, P = 0.479; TACE-SOR vs TACE-JD, P < 0.001; TACE-JD vs TACE, P < 0.001). Independent predictors of OS were treatment allocation, Child-Pugh class large tumor, albumin and extrahepatic metastasis. These findings show that patients with unresectable HCC who were administered TACE-JD survived significantly longer compared with those administered TACE or TACE-SOR.

We explored the hypothesis that sorafenib may improve the effect of transarterial chemoembolization (TACE) in patients with recurrent hepatocellular carcinoma (HCC) and that longer sorafenib duration was associated with additional survival benefits. In this retrospective, nested case-controlled study, 1126 cases of unresectable HCC were collected. Patients with unresectable disease treated with TACE+sorafenib (n=245) and TACE alone (n=245) and those with recurrence after surgery treated with TACE+sorafenib (n=127) and TACE alone (n=127) were identified and matched according to sex, age, and lesion size and number. The clinicopathological factors associated with survival were examined by univariate and multivariate analyses. The mean duration of sorafenib treatment was 10.8±10.51 months. Sorafenib significantly increased the median survival time as compared to TACE alone (unresectable HCC: 20.23 vs. 13.97 months, respectively; p=0.013 and recurrent HCC: 30.7 and 18.22 months, respectively; p=0.003). The survival of patients with unresectable HCC was associated with the presence of portal vein tumor thrombus (HR=1.47, p=0.004) and treatment method (TACE+sorafenib combination therapy; HR=0.72, p=0.003). For patients with recurrent HCC, the presence of extrahepatic metastasis (HR=1.71, p=0.012) and treatment method (TACE+sorafenib therapy; HR=0.60, p=0.002) also was associated with survival. For patients treated with TACE+sorafenib, multivariate analysis showed decreased hazard of death with longer duration of sorafenib treatment (HR=0.9, p<0.001). Thus, sorafenib plus TACE may provide survival benefits, which may be related with sorafenib treatment duration, particularly for patients with HCC recurrence. Further clinical studies are required to confirm these results and identify which patients are most likely to benefit from this therapeutic strategy.

Objectives: To develop prognostic nomograms for patients undergoing hepatectomy for multiple hepatocellular carcinomas (mHCCs)., Background: The prognostic prediction after hepatectomy for mHCCs has not been well established., Methods: A training cohort (n = 540) was analyzed to construct 2 nomograms based separately on data obtained before and after hepatectomy for mHCCs at the Eastern Hepatobiliary Surgery Hospital between 2000 and 2006. The internal and external validations were performed in 2 independent cohorts (n = 180 each) collected from the Eastern Hepatobiliary Surgery Hospital between 2007 and 2010 and the Sun Yat-Sen University between 2000 and 2007. The predictive accuracy was measured by concordance index (C-index) and calibration curve., Results: Serum α-fetoprotein level, hepatitis B virus deoxyribonucleic acid load, end-stage liver disease score, tumor number, total tumor diameter, and the ratio of largest to smallest tumor diameter were incorporated into the preoperative nomogram for overall survival (OS) prediction. In addition to these variables, microvascular invasion, tumor capsule, type of hepatectomy, and local invasion/metastasis were incorporated into the postoperative nomogram. All calibration curves for probability of OS fitted well. In the training cohort, the preoperative nomogram achieved a C-index of 0.75 (95% confidence interval, 0.72-0.78) in predicting OS and accurately stratified patients into 4 prognostic subgroups (5-year OS rates: 65.9%, 46.3%, 29.6%, and 4.1%, P < 0.001). The postoperative nomogram had a C-index of 0.80, which was higher than those of the 4 conventional staging systems (0.53-0.62). These results were supported by the internal and external validations., Conclusions: The 2 nomograms showed accurate pre- and postoperative prediction of posthepatectomy prognosis in patients with mHCCs.

Purpose: This study aimed to establish an effective prognostic nomogram for intrahepatic cholangiocarcinoma (ICC) after partial hepatectomy., Patients and Methods: The nomogram was based on a retrospectively study on 367 patients who underwent partial hepatectomy for ICC at the Eastern Hepatobiliary Surgery Hospital from 2002 to 2007. The predictive accuracy and discriminative ability of the nomogram were determined by concordance index (C-index) and calibration curve and compared with five currently used staging systems on ICC. The results were validated using bootstrap resampling and a prospective study on 82 patients operated on from 2007 to 2008 at the same institution., Results: On multivariate analysis of the primary cohort, independent factors for survival were serum carcinoembryonic antigen, CA 19-9, tumor diameter and number, vascular invasion, lymph node metastasis, direct invasion, and local extrahepatic metastasis, which were all selected into the nomogram. The calibration curve for probability of survival showed good agreement between prediction by nomogram and actual observation. The C-index of the nomogram for predicting survival was 0.74 (95% CI, 0.71 to 0.77), which was statistically higher than the C-index values of the following systems: American Joint Committee on Cancer (AJCC) seventh edition (0.65), AJCC sixth edition (0.65), Nathan (0.64), Liver Cancer Study Group of Japan (0.64), and Okabayashi (0.67; P < .001 for all). It was also higher (0.74) in predicting survival for the mass-forming type of ICC (P < .001). In the validation cohort, the nomogram discrimination was superior to the five other staging systems (C-index: 0.75 v 0.60 to 0.63; P < .001 for all)., Conclusion: The proposed nomogram resulted in more-accurate prognostic prediction for patients with ICC after partial hepatectomy.

Rat ovarian follicle culture, as a novel bioassay, is adopted in this study to explore the effects of cadmium chloride (CdCL(2)) on folliculogenesis and oocyte maturation in vitro; the feasibility for its application on detection of possible effects of chemicals on reproduction is discussed and evaluated as well. The results showed that follicle growth, differentiation, and steroidogenesis were significantly disturbed by ≥ 1.2 μg/mL CdCl(2). The germinal vesicle breakdown of oocyte was also disturbed dose-dependently after the culture follicles were exposed to ≥ 1.6 μg/mL CdCl(2). Exposure to CdCl(2) with concentrations of 1.6 μg/mL on day 2 had caused significant reduced (p < 0.05) survival rate and rate of antral follicles, and increased abnormal follicle rate significantly, compared to the group exposed on day 6. Rat preantral follicle culture is a potential tool to assess the hazards of chemical compounds on female fertility and can be used to elucidate their mechanisms of actions.

Methods for knocking out genes and generating transgenic animals are available to study gene functions during embryogenesis. Recently, RNA interference (RNAi), a conserved eukaryotic mechanism in which double-stranded RNA induces sequence-specific degradation of homologous mRNAs, has become a powerful tool for investigating gene function in cells, whole animals, and embryos. Additionally, in vitro mouse limb bud culture is available to study limb development. By microinjection, we established a new method combining the use of RNAi strategies with in vitro mouse limb bud culture to investigate the functions of various genes during embryonic limb development. Using this new method, we found that HspB10 may play a role during skeletal development of limbs. It shows that the combined use of RNAi strategies (via microinjection) with in vitro mouse limb bud culture system may become a technique for investigating gene function in limb development.