총수담관 결찰에 의한 세담관 변화와 담즙 역류경로에 대한 연구

Abstract

[한글]

임상적으로나 실험적으로 유발된 폐쇄성 황달시 혈청내 direct bilirubin을 포함하여 담적소가 증가되는데 어떤 경로를 통하여 세담관으로부터 혈액내로 유리되는가에 대하여 많은 연구가 진행되고 있으나 아직까지 논란의 대상이 되고 있다. Rich (1930), Rouiller (1956)와 Schaffner 및 Popper (1959)는 정상백서 간세표에서 세담관과 Disse씨 공간사이에 직접적인 연결이 있다고 보고하면서 폐쇄성 황달시 혈청내 담적소가 증가되는 것은 세담관이 파열되면서 세포간 간격을 통하여 담즙이 이동되어 Disse씨 공간을 거쳐 동상혈관으로 나간다고 주장하였다.

그러나 Biava (1964b), Burkel 및 Low (1966)는 전자현미경을 통하여 세담관을 관찰한 결과 정상상태 뿐만 아니라 실험적으로 총수담관을 결찰하여 세담관내 압력을 증가시켰을 때도 세담관에 연해있는 "tight junction"이 파열되지 않고 투과성도 보이지 않아 담즙이 세포간 간격을 통하여 Disse씨 공간으로 나간다는 사실을 부정하였다. Hampton (1958, 1961)은 세담관을 결찰하고 총수담관을 통하여 mercuric sulfide와 thorotrast를 주입한 후 전자현미경으로 이 입자의 이동을 관찰하였더니 간세포에서 pinocytosis에 의하여 간세포로 들어간 후 동상혈관으로 나가는 것을 관찰하고 간세포내에 있는 에너지 소비과정을 통해 능동적으로 pinocytosis가 일어날 것이라고 주장하였다. 이런 사실로 미루어 대부분의 학자들은 세담관 주위에 "tight junction"이 있어 정상상태나 폐쇄성 황달이 있을 때 담즙이 세포간 간격을 통하여 동상혈관으로 나간다는 사실을 부정하고 있다.

그러나 DePalma 및 그의 공동연구자(1968)가 연(鉛)입자를 총수담관을 통하여 주입시켜 보았더니 세포가 간격에 전자밀도가 높은 연(鉛)입자가 출현되는 것을 관찰하고 담즙의 이동은 세포간 간격을 통하여 Disse씨 공간으로 나갈 것이라고 보고하였으며 Schatzki(19

69)도 추적물질로 lanthanum을 사용하여 같은 결과를 얻고 담즙이 세포간 간격을 통하여 이동된다고 주장하였다.

한편 Yodaiken (1966)은 연(鉛)입자를 문정맥과 흉부 대동맥을 통하여 주입하여 전자현미경으로 관찰하였더니 연(鉛)입자가 세포간 간격 뿐만 아니라 간세포내 mitochondria와 핵내에도 출현되는 것을 볼 수 있었다고 보고하였다.

이상의 연구보고들을 참작하여 본 연구에서는 총수담관을 결찰하였을 때 초래되는 폐쇄성 황달시의 간실질세포, 세담관 및 소담관의 형태학적 변화를 관찰함은 물론 총수담관 결찰후 총수담과 문정맥을 통하여 철(鐵)을 함유한 추적물질인 ferridex50을 주입시켜 폐쇄성 황달시 담즙이 어떤 경로를 통하여 혈액내로 유리되는지를 규명하고자 혈청내 담적소, alkaline phosphatase측정, 조직화학적, 광학현미경 및 전자현미경적 관찰을 시행하여 다음과 같은 결론을 얻었다.

1. 혈청내 direct bilirubin, indirect bilirubin 및 alkaline phosphatase치는 결찰후 6시간부터 증가하기 시작하여 bilirubin은 48시간에, alkaline phosphatase는 24시간에 최고치에 달하였다가 그후 다소 감소되었으나 실험마지막날인 10일까지 계속 증가된 치를

유지하였다.

2. 총수담관 결찰로 일어나는 광학현미경소견으로는 결찰후 12시간에 소담관 확장과 담관 상피세포의 비대가 나타났고 결찰후 24시간에 소담관 증식을 보였으며 결찰후 5일에는 간소엽내 신생소담관 형성을 과찰할 수 있었다.

3. 총수담관 결찰후의 전자현미경적 소견상으로는 결찰후 12시간에 세담관확장과 소융모의 파괴 및 소실이 나타났고 24시간에는 원형질 외층의 증대를 관찰할 수 있었으며 5일에는 세담관수의 증가를 관찰할 수 있었으나 "tight junction"은 전균에서 건전하였다.

4. 총수담관 결찰후 ferridex를 총수담관과 문정맥내로 주입하여 철(鐵)염색을 시행한 결과 총수담관 주입군에서는 간소엽주변부 간세포내, 소담관 및 세담관내에 철(鐵)이 분포되었고, 문맥을 통하여 주입한 군에서는 중심정맥과 동상혈관 및 Kupffer 세포내에 철(鐵)이 출현하였다.

5. 전자현미경적 소견으로는 총수담관내로 철(鐵)을 주입한 군에서는 세담관내와 세담관에 인접한 간세포질내에 철(鐵)입자가 분포되어 있었고 간세포질내에는 주로 lysosome이나 pinocytotic vesicle내에 집결되어 나타났다.

문맥내로 주입한 군에서는 동상혈관과 Kupffer세포내에 분포되어 있었다. 세포간 간격에는 총수담관이나 문정맥내로 철(鐵)주입군 모두에서 철(鐵)입자의 존재를 볼 수 없었다.

이상의 소견으로 종합하여 보면 총수담관 결찰시 담즙 역류경로는 "tight junction"을 통한 세포간 간격으로 담즙이 이동된다기 보다는 간세포를 통한 pinocytosis에 의하여 담즙이 혈액내로 이동되는 것으로 사료되었다.

[영문]

In obstructive jaundice, levels of direct reacting bilirubin in the blood stream are elevated. Direct reacting bilirubin is normally excreted in the bile. In obstructive jaundice, it is thought that the bilirubin reenters the blood stream after having been conjugated with glucuronic acid by liver cells.

The bile canaliculus is the smallest radicle of the biliary excretory system of the liver. It consists of a space between two or three liver cells and is bounded by the cell membranes of the liver cells and the points at which the liver cell membranes come together. The points of approximation are generally considered to be impermeable under normal circumstances(Biava, 1964b) and in animals with biliary obstruction as well (Sasaki et al., 1967), and are therefore called "tight junction".

One path that direct reacting bilirubin could take to reenter the blood stream would be directly from the bile canaliculus to the sinusoid, crossing the "tight junction". Previous studies have been performed in an attempt to demonstrate passage of various substances across the "tight junction". Hampton (1958, 1961) injected mercuric sulfide or thorotrast into the common bile duct after ligation and studied sections with electron microscope. He found that the injected particles were taken up at the biliary surface, transported through the cytoplasm, and

discharged at the plasma surface of the parenchymal cells. There was no evidence of passage across the "tight junction".

DePalma et al. (1968) and Schatzki (1969) injected lead acetate solution and lanthanum salt, respectively into the common bile duct or portal vein of rats. In electron micrographs of the liver, they found electron-opaque deposits in the canaliculus, intercellular space, and sinusoid, and found in some electron

micrographs suggestions of passage of the tracer materials across the "tight junction". These studies supported the concept that bile components might gain access to the blood by direct passage between the cells after biliary obstruction.

Yodaiken(1966) injected gum acasia impregnated with lead salts into the portal vein or aorta of rats. In electron micrographs of the liver, he found electron dense lead deposits in the bile canaliculi and sinusoids, and the dense granules were frequently seen in liver mitochondria and also I nuclei.

The present experiment was undertaken in an attempt to study the regurgitation route of bilirubin after common bile duct ligation using an iron containing tracer, ferridex 50, injected into the common bile duct or portal vein of rats. Determination of the regurgitation route of bilirubin after common bile duct

ligation was performed by histologic, histochemical and electron microscopic observations. Concurrently, an assay of direct and indirect bilirubin, and alkaline phosphatase of the blood was carried out. In addition, the general morphologic changes of liver cells and biliary systems in extrahepatic obstructive jaundice following bile duct ligation were observed.

Materials and Methods.

Male albino rats weighing around 200gm were used for the experiment and divided into four groups and treated as follows;

GroupⅠ: Sham operation………………………………………… 12 rats

GroupⅡ: Common bile duct ligation only…………………… 24 rats

GroupⅢ: Ferridex injected, 3 days after bile duct ligation …………18 rats

a; Injected into the common bile duct……………………… 9 rats

b; Injected into the portal vein………………………………9 rats

GroupⅣ; Ferridex injected, 7 days after bile duct ligation …………… 18 rats

a; Injected into the common bile duct……………………… 9 rats

b; Injected into the portal vein………………………………9 rats

The common bile duct was ligated in 60 rats and sham operation in 12 animals under I.V. seconal anesthesia.

In the first series, animals with common bile duck ligation only(GroupⅡ) the rats were sacrificed 6 hours to 10 days following surgery.

In the second series (GroupⅢa and Ⅳa), the common bile ducts of 9 rats were injected with 0.5ml of ferridex 50 solution 3 and 7 days following common bile duct ligation. Three animals from each group were sacrificed at 5, 10 and 15 minutes after ferridex injection.

In the third series(GroupⅢb and Ⅳb), 0.5ml of ferridex solution was injected in 9 rats, via the portal vein 3 and 7 days after common bile duct ligation, respectively. Three animals from each group were sacrificed at 5, 10 and 15 minutes after ferridex injection.

The direct bilirubin, indirect bilirubin and alkaline phosphatase in serum were determined at the time the animals were killed.

Overall histologic alterations were observed by routine hematoxylin-eosin staining technique. In addition, liver sections were stained with Gomori's iron stain for demonstration of the iron distribution in GroupⅢ and Ⅳ.

For electron microscopic examinations, the liver tissue was cut in 1mm cubes, fixed in 1% osmium tetroxide in phosphate buffer at pH 7.4, and embedded in Epon 812. The sections were cut in 400 to 500A thick sections with a glass knife and stained with uranyl acetate and lead hydroxide. Observations were made with a Hitachi HU-11E model electron microscope.

Results and Summary

The liver of rats subjected to ligation of the common bile duct and to retrograde intrabiliary and portal vein injection of an 8iron containing tracer (ferridex) was studied with histologic, histochemical and electron microscopic methods in an attempt to determine the route by which bile reaches plasma under conditions of extrahepatic biliary obstruction. The results are summarized as follows;

1. Serum levels of direct bilirubin, indirect bilirubin and alkaline phosphatase were elevated 6 hours after bile duct ligation. Bilirubin reached a maximum concentration in 48 hours and alkaline phosphatase in 24 hours. After reaching the maximum concentration, the values decreased slightly, but maintained a plateau until the end of experiment, 10 days following duct ligation.

2. Light microscopic examination following bile duct ligation showed that there was dilatation of bile ductules and enlargement of dutular epithelial cells in 12 hours, ductules proliferation in 24 hours, and formation of intralobular bile ductules in 5 days after the liagtion.

3. Electron microscopic findings were dilatation of the bile canaliculi, distortion and loss of microvilli in 12 hours. The pericanalicular zone or ectoplasm was conspicuously widened in 24 hours and there was an increased number of bile canaliculi in 5 days after bile duct ligation. The "tight jundtion" appeared intact.

4. By iron stain in animals injected with ferridex via the common bile duct, iron deposits were seen in the portal tracts, hepatic parenchymal cells in periportal areas and in the dilated bile canaliculi. In animals injected with ferridex via the portal vein, iron deposits were seen in sinusoids and occasionally in Kupffer cells.

5. Under electron microscopic observations, in animals injected with ferriex via the common bile duct, irregular shaped and sized electron dense iron particles were seen in dilated bile canaliculi, lysosomes, and pinocytotic vesicles of hepatic parenhymal cells. In animals injected with ferridex via the portal vein, iron particles were seen in sinusoids and Kupffer cells. The "tight junction" in animals injected via the common bile duct or portal vein appeared intact. No evidence of iron particles within the intercellular space was seen.

In summary, the results of this investigation indicate that in the early stage of obstructive jaundice bile is transported across parenchymal cell cytoplasm from the bile canaliculi to sinusoids rather than through the intercellular spaces opened up

by rupture or increased permeability of the "tight junction" of the bile canaliculi.