[Correlation between Methylation of SALL3 and Cervical Cancer].

Objective To detect the methylation status of SALL3 gene promoter region in normal cervical tissues,cervical cancer tissues,and cervical cancer cell lines and thus explore the relationship between methylation status and the expression of SALL3 gene.Methods The DNA methylation statuses of SALL3 gene in normal cervical,cervical cancer tissues and cervical cancer cell lines were analyzed by methylation-specific PCR(MS-PCR).The expressions of SALL3 mRNA in cervical cancer cell lines,cervical cancer tissues,and normal cervical tissues were detected by RT-PCR.Results In cervical cancer and matched peri-carcinomatous samples,the methylation levels of SALL3 were up-regulated(CCa vs.CCap:P=0.046;CCa vs.NC P=0.039)and the protein expressions were down-regulated(CCa vs.CCap:P=0.012;CCa vs.NC P=0.000)when compared with normal cervix samples.The mRNA levels of SALL3 in HeLa and SiHa cells treated with 5-Azacytidine were elevated in a dose-dependent manner(HeLa:P=0.001;SiHa:P=0.002).The methylation level of SALL3 was higher in high risk human papillomavirus(HPV)-positive cervical samples than in HPV-negative cervical samples(P=0.014),which also resulted in a descending SALL3 expression in HPV-positive samples(P=0.021).Conclusions The hypermethylation of SALL3 in promoter regions inhibits the expression of SALL3 in cervical cancer tissue samples.Infection with high-risk HPV serotypes may increase the methylation of SALL3 promoter region,silence its expression,and thus promote the development of cervical cancer.

Influence of the Intensity, Components, and Spreading of the Deqi Sensation on the Analgesic Effect of SP6 Needling in Primary Dysmenorrhea Patients: A Secondary Analysis of a Randomised Controlled Trial.

Although deqi, the phenomenon whereby excitation of Qi in the meridians occurs with needling, is critical to the practice of acupuncture and its efficacy, it is poorly understood. So we investigate the influence of the deqi sensation on the analgesic effects of acupuncture in patients who were enrolled in a randomised controlled trial for the treatment of patients with primary dysmenorrhea, a painful and common condition, and cold and dampness stagnation. Two groups were assessed: a deqi group (undergoing deep needling with thick needles and manipulation, n=17) and a non-deqi group (undergoing shallow needling with thin needles and no manipulation, n=51). The Sanyinjiao (SP6) was needled for 30 min in both groups. Pain scores at baseline, upon needle removal, and at 10, 20, and 30 min after needle removal were evaluated by the Visual Analogue Scale for pain. The deqi sensation was evaluated by the Acupuncture Deqi Clinical Assessment Scale. Patients who experienced a genuine deqi sensation (n=39) were selected for further analysis. Compared with patients in the non-deqi group who experienced deqi (n=25), patients who self-reported deqi in the deqi group (n=14) felt a stronger deqi sensation, experienced soreness and fullness more frequently, felt a greater intensity of soreness, fullness, electric sensation, spreading, and radiating, and experienced larger spreading distances. In those who experienced the deqi sensation in the deqi group, the intensity of the sensation, as well as their degree of soreness and fullness, was negatively correlated with pain reduction. In patients who experienced the deqi sensation in the non-deqi group, deqi intensity was positively correlated with pain reduction, while soreness was negatively correlated with pain reduction. The distance of spreading was not correlated with pain reduction in either group. We found, in SP6 needling of patients with primary dysmenorrhea with cold and dampness stagnation, that a moderate deqi response predicted a prolonged analgesic effect better than a strong deqi response.

[Analgesic Effect of Deqi Induced by Needling at Sanyinjiao (SP 6) Acupoint on Primary Dysmenorrheal Patients with Cold Damp Stagnation Syndrome].

OBJECTIVE: To observe the analgesic effect of deqi induced by needling at Sanyinjiao (SP 6) on primary dysmenorrheal (PD) patients with cold damp stagnation syndrome (CDSS). METHODS: A total of 64 PD patients with CDSS experiencing abdominal pain (≥40 mm in visual analogue scale ，VAS) were randomly assigned into deqi-expectation(DE) group(n＝15) and no-deqi-expectation(NDE) group(n＝49). On the first day of abdominal pain attack, bilateral SP 6 were punctured respectively with thicker needles with deeper insertion for deqi-expectation patients and thin filiform needles with shallow insertion for no-deqi-expectation patients. The needles were removed after 30 minutes, a deqi scale was used to evaluate the deqi condition. According to the results, patients in the DE group were further divided into deqi DE group and no-deqi DE group, patients in the NDE group were also divided into deqi NDE group and no-deqi NDE group. The VAS was used to evaluate the patients' abdominal pain severity before treatment and 0, 10, 20, 30 min after acupuncture needle withdrawal. RESULTS: The rate of deqi in the DE group was higher than that in the NDE group(P<0.05). The VAS scores of abdominal pain in the four groups were decreased at all time-points after needle withdrawal compared with those before treatment (P<0.01), while the VAS score in the deqi DE group were lower than in the no-deqi NDE group 30 min after needle withdrawal (P<0.05). CONCLUSION: The intervention method of thick needle, deep insertion and some manipulation is easier in inducing deqi than that of thin needle, shallow insertion and no manipulation. The analgesic effect of deqi is better than that of no-deqi for PD patients with CDSS.

Influence of de qi on the immediate analgesic effect of SP6 acupuncture in patients with primary dysmenorrhoea and cold and dampness stagnation: a multicentre randomised controlled trial.

OBJECTIVE: The aim of this multicentre randomised controlled trial was to investigate the contribution of de qi to the immediate analgesic effect of acupuncture in patients with primary dysmenorrhoea and the specific traditional Chinese medicine diagnosis cold and dampness stagnation. METHOD: Eighty-eight patients with primary dysmenorrhoea and cold and dampness stagnation were randomly assigned to de qi (n=43) or no de qi (n=45) groups and underwent 30 min of SP6 acupuncture. The de qi group received deep needling at SP6 with manipulation using thick needles; the no de qi group received shallow needling with no manipulation using thin needles. In both groups the pain scores and actual de qi sensation were evaluated using a visual analogue scale for pain (VAS-P) and the acupuncture de qi clinical assessment scale (ADCAS), respectively. RESULTS: Both groups showed reductions in VAS-P, with no signficant differences between groups. ADCAS scores showed 43/43 and 25/45 patients in de qi and no de qi groups, respectively, actually experienced de qi sensation. Independent of original group allocation, VAS-P reductions associated with actual de qi (n=68) were greater than those without (28.4±18.19 mm vs 14.6±12.28 mm, p=0.008). CONCLUSIONS: This study showed no significant difference in VAS-P scores in patients with primary dysmenorrhoea and cold and dampness stagnation immediately after SP6 acupuncture designed to induce or avoid de qi sensation. Both treatments significantly reduced VAS-P relative to baseline. Irrespective of group allocation, patients experiencing actual de qi sensation demonstrated larger reductions in pain score relative to those without, suggesting greater analgesic effects. TRIAL REGISTRATION NUMBER: Chinese Clinical Trial Registry (ChiCTR-TRC-13003086); Results.

Aberrant Expressions of Immune Factors Facilitate the Disequilibrium of Immune Status in Cervical Cancer.

Objective To explore the expressions and co-relationship of immune factors forkhead box p3 (FoxP3),chemokine (C-C motif) ligand 22 (CCL22),tumor necrosis factor receptor superfamily member 40(OX40),and SMAD family member 3 (Smad3) in cervical carcinoma and investigate their immunomodulatory roles in cervical carcinogenesis.Methods Totally 30 cases of cervical carcinoma with adjacent tissues and 20 cases of normal cervix were collected in this study. FoxP3,CCL22,OX40,and Smad3 mRNA expressions were detected by real-time polymerase chain reaction (RT-PCR). Results Compared to normal cervix,the expression levels of FoxP3 and CCL22 mRNA were elevated in neoplastic foci(P=0.000,P=0.002) and tumor periphery (P=0.048,P=0.040).The mRNAs increased modestly in high-grade squamous cell carcinoma focal(P=0.019,P=0.020) and periphery tissue (P=0.023,P=0.031) in comparison with low-grade squamous cell carcinoma. The expression levels of OX40 and Smad3 mRNA were significantly lower in neoplastic foci(P=0.000,P=0.015) than normal cervix. Compared to low-grade squamous cell carcinoma focal and periphery tissue,the mRNAs decreased moderately in high-grade squamous cell carcinoma(P=0.018,P=0.030; P=0.027,P=0.014). In both neoplastic foci and tumor periphery,the mRNA expression level of CCL22 was positively correlated with FoxP3 (r=0.353,P=0.000; r=0.307,P=0.000) but negatively correlated with OX40 (r=-0.288,P=0.031; r=-0.263,P=0.037),while OX40 was positively correlated with Smad3 (r=0.384,P=0.002;r=0.288,P=0.023). The mRNA expressions of FoxP3 and CCL22 were increased in foci and pericarcinous tissues (P=0.024,P=0.039; P=0.032,P=0.034) while Smad3 was decreased in neoplastic foci (P=0.017) in contrast to HPV negative corresponding group. Conclusion FoxP3 and CCL22 expressions increase while OX40 and Smad3 expression decrease at mRNA level in the microenvironment of cervical cancer,which may be associated with such immunological model that the immunosuppressive roles of FoxP3 and CCL22 enhance while the immunity-boosting roles of OX40 and Smad3 are impeded,contributing to the deterioration of immune disequilibrium in local site and cervical cancer carcinogenesis.

[Our viewpoints on Deqi in the later ages after birth of classical works "The Yellow Emperor's Internal Classic" and "Canon of Difficult Medical Problems"].

In our previous paper, we analyzed "Deqi" in book Huangdi Neijing (The Yellow Emperor's Internal Classic) and Nanjing (Canon of Difficult Medical Problems) from "Zhishen"(Treating mentality) and Tiaoqi (Regulating qi). In the present paper, the authors discuss the connotations of "Deqi" and related events in the later ages of the abovementioned two classic books to the later stage of the Qing Dynasty when involves about 20 classical works as Zhenjiu Dacheng ( The Great Compendium of Acupuncture and Moxibustion), Zhenjing Zhinan (Guide to the classics of Acupuncture), Zhenjiu Daquan (A Complete Works of Acupuncture and Moxibustion) etc. from 1) close association between "Deqi" and patients' mental activity; 2) how to wait for arrival of qi if the needling does not induce "Deqi" for the time being; 3) how to identify "qi-arrival" and then, performing suitable manipulations; 4) Deqi and shallow- or deep-needling; 5) putting more emphasis on patients' feeling and reactions, rather than the practitioners perception beneath the needle which is described in book Huangdi Neijing; and 6) not withdrawing the acupuncture needles if qi does not arrive. Generally, in the later ages, the connotations of Deqi are enriched greatly.

Antitumor activity of endogenous mFlt4 displayed on a T4 phage nanoparticle surface.

AIM: Flt4 plays a key role in promoting tumor metastasis by stimulating solid tumor lymphangiogenesis. In this study, mouse Flt4 (mFlt4) was displayed on T4 phage in order to explore the feasibility of breaking immune tolerance to "self-antigens" and to evaluate the phage's antitumor activity. METHODS: A T4 phage nanometer particle expressing mFlt4 on the surface was constructed for evaluation as a recombinant vaccine. The presence of the mFlt4 gene in the T4-mFlt4 recombinant vaccine was verified by PCR and Western blot analysis. The immunotherapeutic potential of T4-mFlt4 was tested in mice injected with Lewis lung carcinoma (LLC) cells. Anti-Flt4 antibody producing B cells were detected by ELISPOT. The effects of T4-mFlt4 on lymphatic metastasis and lymphangiogenesis were investigated in a mouse antimetastasis assay and by Flt4 and CD105 immunohistochemistry. RESULTS: The T4-mFlt4 recombinant vaccine demonstrated antitumor activity and elicited autoantibodies against mFlt4. Mice carrying LLC-derived tumors exhibited prolonged survival when given the vaccine compared with control-treated animals. The vaccine also inhibited lymphangiogenesis and tumor metastasis in the mouse models. However, T4-mFlt4 was not observed to inhibit tumor growth. CONCLUSION: The T4-mFlt4 recombinant vaccine induced protective antitumor immunity and antimetastasis against LLC. Induction of an autoimmune response directed against tumor progression merits further study as a new strategy for immunotherapy in cancer.