| Background and AimsIt is commonly accepted that there are four certain phases during the process of chronic HBV infection (CHB). They are the phases of immune tolerance, immune clearance, non-active status and active status. It is widely accepted that this four pheases theory is closely associated with Intracellular Distribution of HBcAg. And HBcAg distribution is related to the state of illness, HBV DNA load and response to therapy and also influences the process and improvement of CHB. In clinical practice, it is used to determine HBcAg distribution and expression by following the personal experience of pathologists. However, subjective bias is popular due to the pathological section in less accuracy and self-limitation of pathologists. At present, certain agreement existed in clinical significance of HBcAg distribution, but research conclusions in argument still remain when this topic goes into further step. It is considered that there are three reasons:one is the less objective evaluation towards the distribution and expression of HBcAg. Secondly, there is no standardized and accepted method available in clinical research. Finally, the cases in selections are in huge difference. In addition, it is worthy to discuss whether the "fu-xie" theory in TCM suit the intracellular Distribution of HBcAg and whether the traditional therapy majored in "bu-shen" can be convinced by its effect.Following the above-mentioned, there are four purposes during the research. First of all, to discuss the theoretical possibility that "fu-xie" theory in TCM can be mixed with HBcAg distribution together. Secondly, to measure the intracellular distribution of HBcAg through designing an objective and quantitive method named "sub-class division" and exam its verification. Thirdly, to discuss the clinical significance when the intracellular distribution of HBcAg in different types. Finally, to discuss the intracellular distribution of HBcAg and virological and immune markers under the TCM therapy named the "bu-shen".Methods1. Material resources:429cases of patients in total were obtained from "a study for syndromes regularity of Chronic Hepatitis B virus carriers and treatment of Chinese Herbal formulas", this national technology project was held by professor Zhou Da Qiao.2. Pathological section in imaging and imaging process:Immuno-histochemical staining slices were gathered from Shenzhen Hospital of TCM and photoed in the Shenzhen’s third hospital, then we determined the positive staining condition in HBcAg distribution, numbered and marked the photoed section. Then Image-Pro-Plus imaging software was applied to calculate the numbers of positive cell nucleus in each different field of a photoed section, staining area, mean optical density value (OD value) and mean OD value in the whole field background. The result of the expression of HBcAg in cytoplasm and nucleus in positive respectively was on the basis of chromaticity parameters, and the data of which could be received.3."Sub-class division" method to HBcAg distribution:positive staining rate of HBcAg in cytoplasm and nucleus were calculated according to the relevant formula and the rate of HBcAg were divided into different classes quantitatively according to the data distribution of the rate. Meanwhile, we discuss the relationship between general situation, pathological, virological, immunological markers and HBcAg distribution in order to determine clinical significance of HBcAg distribution.4. serum index:serum HBV DNA was tested by PCR method with COB AS Ampliprep TaqMan48system of Hoffmann-La Roche and one time at initial treatment, in24,52weeks respectively. Serum HBV indexes HBsAg, HBsAb, HBeAg, HBeAb, HBcAb were tested by Abott Laboratories ARCHITECT i2000sr system through Immuno-histochemical staining method.5."bu-shen"therapy effect:This therapy is majored in "bu-shen","jian-pi"and "qing-tou" were attached."Bu-shen-jian-pi" decoction included xianlingpi30g, tusizi10g, duzhongl5g, huainiuqi15g, gouqizi15g, huangqil5g, baizhul5g, fulingl5g, zhuling10g, zhikelOg, yinduyexiazhul5g, danshen20g, sanqi5g, yujingl5g."Bu-shen-qing-tou" decoction included yinduyexiazhu30g, tusizi lOg, xianlingpi30g, nvzhenzi15g, hanliancao15g, chaihu10g, baishao10g, zhishi10g, taorenlOg, gancao5g, huzhang15g. Both decoctions were twice one day. Both decoctions’effects were evaluated according to the comparative between treatment group versus the comparative group in terms of HBcAg distribution and virological、immunological and pathological indexes. The purpose of it is to evaluate the cure effect of "bu-shen"therapy.Results1. the characteristics and clinical significance of HBcAg distribution(1) HBcAg distribution in cytoplasm and nucleus in429patients:most of the patients were concentrated in cytoplasm in the positive rate of0-2.5%, the increase level of the rate is proportional to the decrease of patients’cases, the rate was high in40%in case study. While, more than40%cases were concentrated in nucleus in the positive rate of0-2.5%, the numbers of cases significantly decrease in the rate of2.5-5%.(2) Sub-class division of cytoplasm and nucleus:the results of HBcAg positive rate in cytoplasm as follows,<0.05%positive rate was defined as0class,it means negative expression of HBcAg;0.05-5%was defined as1class,5-10%as2class,10-20%as3class,>20%as4. the results of HBcAg positive rate in nucleus were:0positive rate was defined as0class, it means negative expression of HBcAg;0-2%was defined as1class,2-10%as2class,10-20%as3class,>20%as4.(3) Sex in HBcAg distribution:no matter male or female, both were statistically insignificant in HBcAg distribution in cytoplasm and nucleus (P>0.05)(4) Different age groups in HBcAg distribution:age group was focused on25-47in nucleus,26-47in cytoplasm.(5) Serum HBsAg:serum HBsAg titer in cytoplasm was obviously higher in0class than in3,4class (P<0.05,0.01), which in1,2class was higher than4class (P<0.05, P<0.05). serum HBsAg titer in nucleus was lower in0class than1,2,3,4class (P<0.05,0.001,0.001,0.001),1class was significantly lower than2,3,4class (P<0.01,<0.05,<0.05)(6) Serum HBeAg:serum HBeAg titer in cytoplasm was statistically significant higher in1,3class than in4(P<0.01,<0.05). serum HBeAg titer in nucleus was lower in0class than1,2,3,4class(P<0.001,0.001,0.001,0.001respectively),1class was lower than3class (P<0.05)(7) Serum HBV DNA:Serum HBV DNA load was statistically insignificant in cytoplasm distribution (P>0.05), while in nucleus it was statistically significant lower in0class than in1,2,3,4class (P<0.01,<0.001,0.001,0.001respectively),1class was lower than2,3,4class (P<0.05,0.01,<0.01)(8) IL-2:IL-2was significantly higher in cytoplasm in0class than in1,2,3,4class (P<0.001,0.001,0.001,0.001respectively),1class was higher than2,4class (P<0.001,<0.01). IL-2was statistically insignificantly in nucleus (P>0.05) (9) IL-4:IL-4was significantly lower in cytoplasm in0class than in1,2,3class (P<0.05,<0.01,<0.001),1,2class were both statistically lower than3class, higher than4class (1class, P<0.001,<0.01;2class, P<0.01,0.01),3class was higher than4(P<0.001).IL-4was higher in2class than1,3in nucleus (P<0.05,<0.05)(10) IL-10:IL-10was higher in0,1class than3,4class in cytoplasm (0class, P<0.001,<0.05;1class, P<0.001,<0.01),2class was lower than1class, but higher than3class (P<0.01,<0.01). IL-10was lower in0class than1,4class (P<0.01,<0.05) in nucleus.(11) IFN-γ:IFN-γ was lower in0class than in1,2,3,4class in cytoplasm (P<0.01,0.01,<0.01,<0.001),2class was lower than4class (P<0.05). IFN-γ was statistically insignificant in nucleus.(12) TNF-α:TNF-α was higher in0class than in1,2,3,4class in cytoplasm (P<0.05,0.001,0.01,0.001),1class was higher than2,4class (P<0.001,0.01),2class was lower than3(P<0.05). TNF-a was lower in0class than in2,3class (P<0.01,0.01) in nucleus,1class was also lower than2,3class (P<0.05,0.01)(13) Liver histological pathology:inflammation grade was proportional to HBcAg in cytoplasm in423patients. GO group in cytoplasm distribution was lower than G1, G2, G3group (P<0.01,0.001,0.01), G2group was higher than G1, G3group. But cytoplasm expression in G2group began to reduce. In nucleus, G2group was higher than the other groups (P>0.05). Fibrosis by stages was statistically insignificant in both cytoplasm and nucleus expression (P>0.05)2. The effect of "bu-shen" therapy to HBcAg expression, pathological、virological and immunological indexes(1) Positive staining rate in cytoplasm and nucleus:the mean value of positive nucleus staining rate reduced1.00with statistical insignificance (P>0.05); while the cytoplasm staining rate raised4.48784with statistical significance (P<0.001).While in the contrast group, the mean value of nucleus one raised1.53with statistical insignificance (P>0.05), the mean value of cytoplasm one raised4.58with statistical significance (P<0.001)(2) Liver histological pathology:inflammation grade and fibrosis by stages didn’t improve with statistical insignificance (P>0.05)(3) HBsAg titer:when compared to pre-treatment in treatment group, the titer of the post-treatment decreased21632.51IU/ml with statistical significance (P<0.01). While compared to the post-treatment in the contrast group, the titer decreased20689.3867IU/ml with statistical insignificance (P>0.05)(4) HBeAg titer:when compared to pre-treatment in treatment group, the titer of the post-treatment decreased134.3548IU/ml with statistical significance (P<0.001).While compared to the post-treatment in the contrast group, the titer decreased115.0866IU/ml, P>0.05(5) HBV DNA load:when compared to pre-treatment in treatment group, HBV DNA index decreased0.5638with statistical significance (P<0.001) in the post-treatment. Being compared to the post-treatment in the contrast group, treatment group decreased0.483with statistical significance (P<0.05)(6)IL-2:it increased22.5121pg/ml with statistical significance (P<0.05) after treating by TCM. Being compared to the post-treatment in the contrast group, the treatment group increased21.3036pg/ml with statistical significance (P<0.05)(7)IL-4:it decreased42.5714pg/ml with statistical significance(P<0.05)after treating by TCM. Being compared to the post-treatment in the contrast group, the treatment group decreased42.0477pg/ml with statistical significance (P<0.01)(8) IL-10:it decreased1.7844pg/ml with statistical insignificance (P>0.05), Being compared to the post-treatment in the contrast group, the treatment group decreased0.962pg/ml with statistical insignificance.(9) IFN-y:it increased1.1039pg/ml with statistical insignificance (P>0.05), Being compared to the post-treatment in the contrast group, the treatment group increased1.6257pg/ml with statistical insignificance.(10) TNF-a:it decreased15.3377pg/ml with statistical significance (P<0.05), Being compared to the post-treatment in the contrast group, the treatment group decreased13.3355pg/ml with statistical significance (P<0.05)Conclusion1."Sub-class division" method is quantified on the basis of data from positive staining of HBcAg in cytoplasm and nucleus. This method is objective to discuss the relationship between intracellular distribution of HBcAg and virological, immunological and pathological indexes.2. Followed the increase of the class level (the level of positive staining rate increased), HBsAg、HBeAg、HBV DNA、IL-10、TNF-α was characterized in nucleus expression, while IL-2, IL-4, IFN-ywas in cytoplasm expression.3."Bu-shen" therapy could raise the level of IL-2and down-regulated the level of IL-4, IL-10, TNF-α. The therapy regulated the immune status of the body, reduced HBsAg, HBeAg titer and HBV DNA load. This therapy could improve the condition of immune tolerance in patients, it could change the fact that HBV virus went on damaging the liver cell, and decrease of HBV DNA load. 4. Since the positive staining rate of HBcAg in cytoplasm was proportional to inflammation grade, the positive staining rate of HBcAg in nucleus was proportional to HBV DNA load, and HBcAg expression in cytoplasm increased after "Bu-shen"therapy was used. So it can be convinced that the evil factor was forced to come out from inside, the clinical feature of fu-xie, equally suited for HBcAg distribution and expression. |
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